Agents for influenza neutralization

ABSTRACT

The present invention provides antibodies (e.g., monoclonal antibodies, human antibodies, humanized antibodies, etc.), which bind to multiple influenza strains. Such antibodies are useful, for example, in the prophylaxis, treatment, diagnosis, and/or study of influenza.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/645,453, filed May 10, 2012, the contentsof which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant number R37GM057073 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “Sequence Listing.txt” onMar. 14, 2013). The .txt file was generated on Mar. 8, 2013 and is 39.3kb in size. The entire contents of the Sequence Listing are hereinincorporated by reference.

BACKGROUND OF THE INVENTION

Influenza virus is a global health threat that is responsible for over300,000 deaths annually. The virus evades immune recognition by engagingin a combination of accelerated antigenic drift, domain reassortment,genetic recombination, and glycosylation based masking of its surfaceglycoproteins. This rapid mutation capability of the virus isparticularly exacerbated in the context of the growing threat from thepresent H1N1 ‘swine flu’ pandemic as well as the alarming worldwidespate in recent infections with highly pathogenic avian H5N1 ‘bird flu’influenza strains. (Khanna et al., Journal of Biosciences, 33(4):475,2008, Soundararajan et al., Nature Biotechnology 27:510, 2009).Furthermore, two of the major flu pandemics of the last centuryoriginated from avian flu viruses that changed their genetic makeup toallow for human infection.

There is a need for the development of effective anti-influenzaprophylactics and therapeutics. Furthermore, given the high degree ofunpredictability in evolution of these influenza viruses, there is aparticular need for the development of cross-strain effective (e.g.,“universal” or “broad spectrum”) anti-influenza prophylactics andtherapeutics. Such effective anti-influenza agents, and particularlysuch universal or broad spectrum anti-influenza agents could replace oraugment vaccines designed to target specific ‘seasonal’ viral strains incirculation (Ekiert et al., Science, 324(5924):246, 2009 and Sui et al.,Nat Struct Mol Biol. 16(3):265, 2009). Alternatively or additionally,there is a need for the development of effective anti-influenzaprophylactics or therapeutics that can replace or augment currentanti-viral therapy. The importance of such agents is highlighted by theemerging drug resistance to current antivirals Tamiflu/Relenza(NA-inhibitors) and Amantadine/Rimantadine (MP-2 inhibitors) (Collins etal., Nature 453:1258, Stouffer et al., Nature, 451:596, 2008, Pielak etal., Proc. Natl. Acad. Sci. USA, 106:7379, 2009). For instance, over 98%and 100% of H1N1 strains in the 2011/2012 flu season are resistant toTamiflu and the adamantane derivatives (Amantadine/Rimantadine),respectively.

SUMMARY

The present invention provides new influenza binding agents. Among otherthings, the present invention provides influenza binding agents thatbind to multiple influenza strains. The present invention specificallyprovides binding agents that bind to influenza hemagglutinin (HA). Thepresent invention particularly provides certain antibodies that bind toinfluenza HA. In some embodiments, such antibodies are characterized bybinding to a particular HA epitope and/or to HA from a group 1 virus, agroup 2 virus or, in some embodiments, both. In some embodiments,provided antibodies bind to an HA selected from the group consisting ofan HA polypeptide of subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, H16, and combinations thereof. In someembodiments, provided antibodies are characterized by an ability toneutralize infection by a group 1 virus, a group 2, virus, or in someembodiments, both. In some such embodiments, such provided antibodiesshow a neutralization IC₅₀ (ug/ml) within a range as described and/orexemplified herein. In some embodiments, such provided antibodies show aneutralization IC₅₀ (ug/ml) whose lower bound is about 0.1 ug/ml andupper bound is about 10 ug/ml. In some embodiments, such providedantibodies show a neutralization IC₅₀ (ug/ml) whose lower bound isselected from the group consisting of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or more ug/ml, and whose upper boundis higher than the lower bound and is selected from the group consistingof 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or more ug/ml.

In some embodiments, such provided antibodies show binding to influenzaHA (e.g., group 1 and/or group 2 subtypes) with a K_(D) (nM) less than2000 nM, less than 1500 nM, less than 1000 nM, less than 500 nM, lessthan 250 nM, less than 225 nM, less than 200 nM, less than 175 nM, lessthan 150 nM, less than 125 nM, less than 100 nM, less than 75 nM, orless than 50 nM.

In some embodiments, such provided antibodies show binding to influenzaHA with a K_(a) (M⁻¹s⁻¹) whose lower bound is about 0.01×10⁵ M⁻¹s⁻¹ andupper bound is about 1.0×10⁶ M⁻¹s⁻¹. such provided antibodies showbinding to influenza HA with a K_(a) (M⁻¹s⁻¹) whose lower bound isselected from the group consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵,0.04×10⁵, 0.08×10⁵, 0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵,1.0×10⁵, 1.2×10⁵, 1.4×10⁵, 1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more M⁻¹s⁻¹,and whose upper bound is higher than the lower bound and is selectedfrom the group consisting of 1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵,3.0×10⁵, 3.5×10⁵, 4.5×10⁵, 5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵,7.5×10⁵, 8.0×10⁵, 8.5×10⁵, 9.0×10⁵, 9.5×10, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶,1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or moreM⁻¹s⁻¹.

In some embodiments, such provided antibodies show binding to influenzaHA with a K_(d) (s⁻¹) whose lower bound is about 0.01×10⁵ s⁻¹ and upperbound is about 1.0×10⁶ s⁻¹. such provided antibodies show binding toinfluenza HA with a K_(a) (s⁻¹) whose lower bound is selected from thegroup consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵, 0.04×10⁵, 0.08×10⁵,0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵, 1.0×10⁵, 1.2×10⁵, 1.4×10⁵,1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more s⁻¹, and whose upper bound is higherthan the lower bound and is selected from the group consisting of1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵, 3.0×10⁵, 3.5×10⁵, 4.5×10⁵, 5.0×10⁵,5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵, 7.5×10⁵, 8.0×10⁵, 8.5×10⁵, 9.0×10⁵,9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or more s⁻¹.

The present invention also defines structural features of certainprovided antibodies that confer particular functional attributes (e.g.,HA binding, neutralization, subtype specificity, etc). The presentinvention therefore provides binding agents that share such structuralfeatures, and in some embodiments therefore also functional attributes,of these provided antibodies. For example, in some embodiments, thepresent invention provides binding agents that share structuralfeatures, and functional attributes, of one or more of the certainprovided antibodies having amino acid sequences as set forth in Tables 2and/or 3 herein.

In some embodiments, such structural features of such certain providedantibodies include one or more CDRs or one or more FRs at least 80%identical in sequence to a corresponding CDR or FR from Tables 2 and 3(SEQ ID NO: 1-60). In some embodiments, such structural features of suchcertain provided antibodies include one or more CDRs and/or one or moreFRs that is identical in sequence to a corresponding CDR or FR fromTables 2 and 3 (SEQ ID NO: 1-60). In some embodiments, such structuralfeatures of such certain provided antibodies include CDRs and FRs thatare identical in sequence to those set forth in Tables 2 and 3 (SEQ IDNO: 1-60).

In some embodiments, such structural features include CDR and FRsequence elements, each of which is identical to a reference CDR or FRsequence element set forth in Table 2 and/or Table 3 (SEQ ID NOs:1-60)except that it includes one or more amino acid substitutions withrespect to that reference sequence element, where the included CDR andFR sequence elements together contain no more than 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in CDR and FRsequences, as compared with the corresponding CDR and FR referencesequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). In someembodiments, such structural features include CDR and FR sequenceelements together contain no more than 18 substitutions as compared withthe corresponding CDR and FR reference sequence elements from Tables 2and 3 (SEQ ID NO: 1-60). In some embodiments, such structural featuresinclude CDR and FR sequence elements together contain no more than 15substitutions as compared with the corresponding CDR and FR referencesequence elements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features includes a FR sequenceelement, which is identical to a reference FR sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included FR sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in FR sequence, as compared with the corresponding FRreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features includes a CDR sequenceelement, which is identical to a reference CDR sequence element setforth in Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that itincludes one or more amino acid substitutions with respect to thatreference sequence element, where the included CDR sequence elementcontains no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 substitutions in CDR sequence, as compared with thecorresponding CDR reference sequence elements from Tables 2 and 3 (SEQID NO: 1-60).

In some embodiments, such structural features includes a VH sequenceelement, which is identical to a reference VH sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included VH sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in CDR sequence, as compared with the corresponding VHreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). Insome embodiments, such structural features include a structural elementcorresponding to any one of VH-1, VH-2, VH-3, VH-4, VH-5, VH-6, VH-7,VH-8, VH-9, VH-10, VH-11, VH-12, VH-13, VH-14, VH-15, VH-16 or fragmentthereof, as compared with the corresponding VH reference sequenceelements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features includes a VL sequenceelement, which is identical to a reference VL sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included VL sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in CDR sequence, as compared with the corresponding VHreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). Insome embodiments, such structural features include a structural elementcorresponding to any one of VL-1, VL-2, VL-3, VL-4, VL-5, VL-6, VL-7,VL-8, VL-9, VL-10, VL-11, or fragment thereof, as compared with thecorresponding VL reference sequence elements from Tables 2 and 3 (SEQ IDNO: 1-60).

In some embodiments, such structural features include a structuralelement corresponding to any one of VH-1, VH-2, VH-3, VH-4, VH-5, VH-6,VH-7, VH-8, VH-9, VH-10, VH-11, VH-12, VH-13, VH-14, VH-15, VH-16 orfragment thereof combined with any one of VL-1, VL-2, VL-3, VL-4, VL-5,VL-6, VL-7, VL-8, VL-9, VL-10, VL-11, or fragment thereof. In someembodiments, VH-1 (SEQ ID NO:1) is combined with VL-1 (SEQ ID NO:33).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR1sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 1 sequence element that has twoor more amino acid substitutions as compared to a reference CDR1sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 1 sequence element that has oneor more amino acid substitutions as compared to reference CDR1 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has at least two aminoacid substitutions as compared to reference CDR1 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR1 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR1 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR2sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 2 sequence element that has twoor more amino acid substitutions as compared to a reference CDR2sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 2 sequence element that has oneor more amino acid substitutions as compared to reference CDR2 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has at least two aminoacid substitutions as compared to reference CDR2 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR2 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR2 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR3sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 3 sequence element that has twoor more amino acid substitutions as compared to a reference CDR3sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 3 sequence element that has oneor more amino acid substitutions as compared to reference CDR3 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has at least two aminoacid substitutions as compared to reference CDR3 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR3 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR3 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a VH frameworkregion sequence element that shows more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 99% percent identity with a reference VH framework regionsequence element from Table 2 (SEQ ID NOs:1-16).

In some embodiments, such structural features include a VL frameworkregion sequence element that shows more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 99% percent identity with a reference VL framework regionsequence element from Table 3 (SEQ ID NOs:33-43).

In some embodiments, the present invention provides a binding agent thatincludes such structural features of such certain provided antibodiessuch that the provided binding agent shares with the certain providedantibodies the functional attribute that it binds to influenza HAselected from the group consisting of group 1 subtype, group 2 subtype,and combinations thereof.

In some embodiments, a provided binding agent shows an influenzaneutralization IC₅₀ (ug/ml) within a range as set forth herein for aparticular provided antibody. In some embodiments, a provided bindingagent shows such an IC₅₀ (ug/ml) for an influenza virus of group 1subtype, group 2 subtype, or both. In some embodiments, a providedbinding agent is characterized by a functional attribute of an influenzaneutralization IC₅₀ (ug/ml) within a range as described and/orexemplified herein. In some embodiments, such provided binding agentshows a neutralization IC₅₀ (ug/ml) whose lower bound is about 0.1 ug/mland upper bound is about 10 ug/ml. In some embodiments, such providedbinding agent shows a neutralization IC₅₀ (ug/ml) whose lower bound isselected from the group consisting of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or more ug/ml, and whose upper boundis higher than the lower bound and is selected from the group consistingof 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or more ug/ml.

In some embodiments, such provided binding agent shows binding toinfluenza HA (e.g., group 1 and/or group 2 subtypes) with a K_(D) (nM)less than 2000 nM, less than 1500 nM, less than 1000 nM, less than 500nM, less than 250 nM, less than 225 nM, less than 200 nM, less than 175nM, less than 150 nM, less than 125 nM, less than 100 nM, less than 75nM, or less than 50 nM.

In some embodiments, such provided binding agent shows binding toinfluenza HA with a K_(a) (M⁻¹s⁻¹) whose lower bound is about 0.01×10⁵M⁻¹s⁻¹ and upper bound is about 1.0×10⁶ M⁻¹s⁻¹. such provided antibodiesshow binding to influenza HA with a K_(a) (M⁻¹s⁻¹) whose lower bound isselected from the group consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵,0.04×10⁵, 0.08×10⁵, 0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵,1.0×10⁵, 1.2×10⁵, 1.4×10⁵, 1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more M⁻¹s⁻¹,and whose upper bound is higher than the lower bound and is selectedfrom the group consisting of 1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵,3.0×10⁵, 3.5×10⁵, 4.5×10⁵, 5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵,7.5×10⁵, 8.0×10⁵, 8.5×10⁵, 9.0×10⁵, 9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶,1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or moreM⁻¹s⁻¹.

In some embodiments, such provided binding agent shows binding toinfluenza HA with a K_(d) (s⁻¹) whose lower bound is about 0.01×10⁵ s⁻¹and upper bound is about 1.0×10⁶ s⁻¹. such provided antibodies showbinding to influenza HA with a K_(a) (s⁻¹) whose lower bound is selectedfrom the group consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵, 0.04×10⁵,0.08×10⁵, 0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵, 1.0×10⁵, 1.2×10⁵,1.4×10⁵, 1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more s⁻¹, and whose upper boundis higher than the lower bound and is selected from the group consistingof 1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵, 3.0×10⁵, 3.5×10⁵, 4.5×10⁵,5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵, 7.5×10⁵, 8.0×10⁵, 8.5×10⁵,9.0×10⁵, 9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶,1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or more s⁻¹.

In some embodiments, the present invention provides a binding agent thatincludes such structural features of such certain provided antibodiessuch that the provided binding agent shares with the certain providedantibodies the functional attribute that it competes with one or more ofthe antibodies listed in Tables 2 and 3 (SEQ ID NO: 1-60) for binding toat least one HA polypeptide. In some embodiments, a provided bindingagent such structural features that it competes with one or more of theantibodies listed in Tables 2 and 3 (SEQ ID NO: 1-60) for binding to aplurality of different HAs, which plurality of different HA polypeptidesincludes HA polypeptides proteins found in at least 2 different HAsubtypes genotypes.

In some embodiments, a provided binding agent is characterized by afunctional attribute of binding to one or more of HA polypeptides ofsubtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and/or H16. In some embodiments, a provided binding agent binds toat least two HA polypeptides of subtype H1, H3, H5, H7, and H9.

In some embodiments, a provided binding agent in characterized by afunctional attribute of binding to one or more epitopes in the MPERregion of an HA polypeptide. In some embodiments, a provided bindingagent is characterized by a functional attribute of binding to one ormore epitopes in the MPER region of an HA polypeptide independent of itsglycosylation. In some embodiments, a provided binding agent ischaracterized by a functional attribute of binding to one or moreepitopes in the HA-1 (head) and/or HA-2 (stalk) domains of an HApolypeptide. In some embodiments, a provided binding agent ischaracterized by a functional attribute of binding to one or moreepitopes located within the HA-1/HA-2 interface membrane proximalepitope region (MPER). In some embodiments, a provided binding agent ischaracterized by a functional attribute of binding to one or moreepitopes located within the canonical α-helix and/or residues in itsvicinity.

In some embodiments, a provided binding agent is or comprises apolypeptide. In some embodiments, a provided binding agent is orcomprises an antibody or fragment thereof. In some embodiments, aprovided binding agent is or comprises a monoclonal antibody or fragmentthereof. In some embodiments, the binding agent is or comprises a “fulllength” antibody, in that it contains two heavy chains and two lightchains, optionally associated by disulfide bonds as occurs withnaturally-produced antibodies. In some embodiments, the binding agent isor comprises a fragment of a full-length antibody in that is containssome, but not all of the sequences found in a full-length antibody. Forexample, in some embodiments, the binding agent is or comprises antibodyfragments which include, but are not limited to, Fab, Fab′, F(ab′)2,scFv, Fv, dsFv diabody, and Fd fragments. In some embodiments, aprovided binding agent is or comprises an antibody that is a member ofan antibody class selected from the group consisting of IgG, IgM, IgA,IgD, IgE or fragment thereof. In some embodiments, a provided bindingagent is or comprises an antibody produced by chemical synthesis. Insome embodiments, a provided binding agent is or comprises an antibodyproduced by a cell. In some embodiments, a provided binding agent is orcomprises a chimeric antibody, for example from mouse, rat, horse, pig,or other species, bearing human constant and/or variable region domains.

In some embodiments, a provided binding agent is or comprises apolypeptide with antibody CDRs. In some embodiments, a provided bindingagent is or comprises a scaffolding domain, such as protein A,lipoclins, ankryin consensus repeat domain, thioredoxin, adnectin,anticalins, centyrin, avimer domains, ubiquitin, zinc finger DNA-bindingproteins (ZEPs), or IgNARs, which is used to displays one or more CDRs.In some embodiments, a provided binding agent is or comprises acystine-knot miniprotein. In some embodiments, a provided binding agentis or comprises an avibody (diabody, tribody, tetrabody). In someembodiments, a provided binding agent is or comprises a Scorpion,wherein the Scorpion structure comprises two binding moieties separatedby an immunoglobulin Fc domain. In some embodiments, a provided bindingagent is or comprises a VHH (i.e., an antigen-specific VHH) antibodythat comprises only a heavy chain. In some embodiments the VHH isderived from a llama or other camelid antibody (e.g., a camelid IgG2 orIgG3, or a CDR-displaying frame from such camelid Ig). In someembodiments such a VHH is derived from a shark.

In some embodiments, a provided binding agent is or comprises one ormore “mini-antibodies” or “minibodies”, which are sFv polypeptide chainsthat include oligomerization domains at their C-termini, separated fromthe sFv by a hinge region. In some embodiments, the hinge regioncomprises a self-associating alpha-helix or leucine zipper, which may ormay not be further stabilized by additional disulfide bonds. In someembodiments, a provided binding agent is or comprises a peptidomimetic.In some embodiments, a provided binding agent is or comprises amimeotope.

In some embodiments, a provided binding agent is or comprises aconjugate, in which a binding agent moiety (comprises or consists of thebinding agent or a functional portion thereof) with a conjugated moiety.In some embodiments such a conjugated moiety is an entity. In someembodiments, such an entity is a chemical class selected from the groupconsisting of polypeptides, carbohydrates, lipids, small organicmolecule, organic polymer, inorganic polymer, metals, ions, isotopes orcombinations thereof. In some embodiments such a conjugated moiety is orcomprises a therapeutic or diagnostic payload. In some embodiments, sucha conjugated moiety is or comprises a detectable payload. In someembodiments, such a conjugated moiety is or comprises a detectableentity. In some embodiments, such a conjugated moiety is or comprises anaffinity agent. In some embodiments, such a conjugated moiety is orcomprises a targeting agent. In some embodiments, such a conjugatedmoiety is or comprises a masking and/or stabilizing agent. In someembodiments, a provided conjugate comprises a single binding agentmoiety and a plurality of conjugated moieties; in some embodiments suchplurality of conjugated moieties comprises a plurality of the sameconjugated moiety; in some embodiments such plurality of conjugatedmoieties includes two or more different conjugated moieties, optionallyof different types (e.g., therapeutic payloads, detectable payload,targeting agent, affinity agent etc.).

In some embodiments, a provided binding agent is or comprises a nucleicacid, such as DNA or RNA. In some embodiments the nucleic acid isdesigned to mimic an epitope within a hemagglutinin (HA) polypeptide. Insome embodiments the nucleic acid is designed to mimic a conservedepitope within one or more Influenza HA polypeptide subtypes. In someembodiments, a provided binding agent is or comprises one or moreoligonuclotides. In some embodiments, a provided binding agent is orcomprises one or more oligonuclotides comprising a secondary structuresuch as loop, hairpin, fold or combinations thereof. In someembodiments, a provided binding agent is or comprises one or moreoligonuclotides comprising a higher ordered (tertiary or quaternary)structure. In some embodiments, a provided binding agent is or comprisesan aptamer.

In some embodiments, the present invention provides a cell or cell lineexpressing a binding agent as described herein. In some embodiments,such a cell or cell line is a mammalian cell or cell line. In certainembodiments, such a cell or cell line is a hybridoma.

In some embodiments, the present invention provides a method of treatinga patient, the method comprising steps of administering to a patientsuffering from or susceptible to influenza infection a compositioncomprising a binding agent described herein, in an appropriate unitdosage form for delivery according to a regimen that correlates withreduction in incidence and/or severity, and/or with delay of onset ofone or more manifestations or effects of influenza infection.

In some embodiments, the present invention provides a kit comprising atleast one binding agent as described herein, formulated foradministration via an administration device, together with such anadministration device in a set comprising one or more containers. Insome embodiments, an appropriate administration device is selected fromthe group consisting of a syringe, needle, applicator, and combinationsthereof. In some embodiments, a provided kit includes instructions foruse.

Provided binding agents, compositions, and methods are useful, forexample, in research and/or in medicine. In some embodiments, providedbinding agents, compositions, and methods are useful, for example, inprophylaxis, treatment, diagnosis, and/or study of influenza. Forexample, in some embodiments, provided binding agents or compositionsare administered to subjects suffering from or susceptible to aninfluenza infection. In some embodiments, provided binding agents orcompositions are administered prior to known exposure to influenza, orto particular influenza subtypes or strains. In some embodiments,provided binding agents are administered after known exposure toinfluenza, or to particular influenza subtypes or strains. In someembodiments, provided binding agents or compositions are administeredprior to manifestation of effects or symptoms of influenza infection, orto one or more particular effects or symptoms of influenza infection,including of influenza infection with one or more particular influenzasubtypes or strains. In some embodiments, provided binding agents orcompositions are administered prior to manifestation of effects orsymptoms of influenza infection, or to one or more particular effects orsymptoms of influenza infection, including influenza infection with oneor more particular influenza subtypes or strains.

In some embodiments, provided binding agents or compositions areadministered to a subject in combination with one or more otheranti-influenza therapies and/or with one or more other therapies for aneffect on symptom of influenza infection (e.g., inflammation, fever,nausea, weight loss, loss of appetite, rapid breathing, increase heartrate, high blood pressure, body aches, muscle pain, eye pain, fatigue,malaise, dry cough, runny nose, and/or sore throat).

BRIEF DESCRIPTION OF THE DRAWING

The Figures described below, that together make up the Drawing, are forillustration purposes only, not for limitation.

FIG. 1 depicts a structural representation of the various parts, regionsand domains of a mammalian antibody.

FIGS. 2A and B depicts HA binding affinity for an exemplary antibody.FIG. 1A (top panel) demonstrates that the particular depicted antibodybinds to both group 1 and group 2 subtypes of HA with differentialbinding affinity; and FIG. 1A (bottom panel) and FIG. 1B show that theantibody binds specifically to HA.

FIGS. 3A-E depicts HA binding affinity and kinetics for an exemplaryantibody over a range of concentrations. The particular antibody bindsto both group 1 and group 2 subtypes of HA with differential bindingaffinity and kinetics.

FIG. 4 depicts HA binding affinity and neutralization for an exemplaryantibody. The particular antibody inhibits PR8 Virus (H1N1) influenzavirus-induced plaque production for 6 different doses.

FIG. 5 presents effects of an exemplary antibody pre-incubation with PR8on infectivity in MDCK cells. After infection, MDCK cells were grown invirus-free media with varying concentration of antibody for 48 hoursbefore viral titer was quantified by real time PCR using primersspecific to the virus to calculate an IC₅₀ value.

FIG. 6 presents data from mice treated with an exemplary antibody in anH1N1 challenge. In this challenge, mice treated with exemplary antibodyhave a lower percent of weight loss post infection compared to untreatedcontrol.

FIG. 7 presents data from a H1N1 challenge in mice. AS can be seen, anexemplary antibody delays the development of H1N1 infection in mice ascompared to that observed with a PBS control. The observed decrease ininfectivity (i.e., in the rate of onset of symptoms of infection) in thepresence of exemplary antibody is comparable to that seen with theantiviral drug Ribavirin.

FIG. 8 presents data from mice treated with an exemplary antibody in anH1N1 challenge. In this challenge, mice treated with an exemplaryantibody post-infection demonstrate a recovery in weight.

FIG. 9 depicts the pharmacokinetic profile observed in mice treated withan exemplary antibody. Following treatment with an exemplary antibody,sera samples were collected over time and evaluated for antibodyconcentration.

FIG. 10A-B shows an alignment of exemplary VH (FIG. 10A) and VL (FIG.10B) sequences. Each exemplary sequences is also shown in Tables 2and/or 3.

DEFINITIONS

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand (e.g., an antibody) binds to itspartner (e.g., an epitope). Affinities can be measured in differentways.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an l-amino acid. “Standardamino acid” refers to any of the twenty standard l-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxy-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, ofeither sex and at any stage of development. In some embodiments,“animal” refers to non-human animals, at any stage of development. Incertain embodiments, the non-human animal is a mammal (e.g., a rodent, amouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, aprimate, and/or a pig). In some embodiments, animals include, but arenot limited to, mammals, birds, reptiles, amphibians, fish, insects,and/or worms. In certain embodiments, the animal is susceptible toinfection by influenza. In some embodiments, an animal may be atransgenic animal, genetically engineered animal, and/or a clone.

Antibody: As used herein, the term “antibody” refers to a polypeptidehaving structural characteristics of an immunoglobulin, as those areunderstood in the art. As produced by mammalian cells in nature, animmunoglobulin has the structure depicted in FIG. 1. A typicalimmunoglobulin (antibody) structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (approximately 25 kD)and one “heavy” chain (approximately 50-70 kD). The N-terminus of eachchain defines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms “variable lightchain” (VL) and “variable heavy chain” (VH) refer to these light andheavy chains respectively. Each variable region is further subdividedinto hypervariable (HV) and framework (FR) regions. The hypervariableregions comprise three areas of hypervariability sequence calledcomplementarity determining regions (CDR 1, CDR 2 and CDR 3), separatedby four framework regions (FR1, FR2, FR2, and FR4) which form abeta-sheet structure and serve as a scaffold to hold the HV regions inposition. The C-terminus of each heavy and light chain defines aconstant region consisting of one domain for the light chain (CL) andthree for the heavy chain (CH1, CH2 and CH3). In many embodiments, anantibody is a polypeptide whose amino acid sequence includes one or morestructural elements recognized by those skilled in the are as acomplementarity determining region (CDR). In some embodiments, anantibody is a polypeptide whose amino acid sequence includes structuralelements recognized by those skilled in the art as an immunoglobulinvariable domain. In some embodiments, an antibody is “full length” inthat in contains two heavy chains and two light chains, optionallyassociated by disulfide bonds as occurs with naturally-producedantibodies. In some embodiments, an antibody is a fragment of afull-length antibody in that it contains some, but not all of thesequences found in a full-length antibody. For example, in someembodiments, Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. Insome embodiments, an antibody is a member an antibody class selectedfrom the group consisting of IgG, IgM, IgA, IgD, and IgE. In someembodiments, an antibody is produced by chemical synthesis. In someembodiments, an antibody is a monoclonal antibody. In some embodiments,an antibody is a polyclonal antibody. In some embodiments, an antibodyis produced by a cell. In some embodiments, an antibody is produced bychemical synthesis. In some embodiments, an antibody is derived from amammal. In some embodiments, an antibody is derived from an animal suchas, but not limited to, mouse, rat, horse, pig, or goat. In someembodiments, an antibody is produced using a recombinant cell culturesystem. In some embodiments an antibody is a chimeric antibody, forexample, from mouse, rat, horse, pig, or other species, bearing humanconstant and/or variable regions domains. In some embodiments, anantibody is a derived from a human. In some embodiments, an antibody isa polyclonal antibody. In some embodiments, an antibody is a humanizedantibody.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Binding agent: As used herein, the term “binding agent” refers to anagent that is capable of binding to an antigen or biological target. Insome embodiments, a binding agent comprises a protein. In someembodiments, a binding agent is or comprises a naturally occurringprotein. In some embodiments, a binding agent is derived from a cell ora virus. In some embodiments, a binding agent is a synthetic orchemically synthesized protein. In some embodiments, binding agents arecomprised of natural amino acids. In other embodiments, a binding agentcomprises one or more unnatural amino acids. In some embodiments, abinding agents is comprised of a combination of natural and unnaturalamino acids. In some embodiments, a binding agent is comprised of one,two or more polypeptide chains that are covalently or non-covalentlyassociated. In some embodiments, a binding agent may be linked to, orpart of, a longer polypeptide chain, so long as the binding agentretains its three-dimensional structure and arrangement for interaction.In some embodiments, a binding agent may be appended to the N- orC-termini of another polypeptide sequence that is or is not a bindingagent. In some embodiments, a binding agent may be incorporated into thesequence of another polypeptide that is or is not a binding agent,thereby separating the polypeptide sequence into two or more segments.

In some embodiments, a binding agent is a protein that functionssimilarly to an antibody and is able to bind to a specific antigen toform a complex and elicit a biological response (e.g., agonize orantagonize a particular biological activity.) In some embodiments, thebinding agent is an antibody. In some embodiments, the binding agent isor comprises a “full length” antibody, in that it contains two heavychains and two light chains, optionally associated by disulfide bonds asoccurs with naturally-produced antibodies. In some embodiments, thebinding agent is or comprises a fragment of a full-length antibody inthat is contains some, but not all of the sequences found in afull-length antibody. For example, in some embodiments, the bindingagent is or comprises antibody fragments which include, but are notlimited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fdfragments. In some embodiments, a provided binding agent is or comprisesa VHH (i.e., an antigen-specific VHH) antibody that comprises only aheavy chain. In some embodiments the VHH is derived from a llama orother camelid antibody (e.g., a camelid IgG2 or IgG3, or aCDR-displaying frame from such camelid Ig). In some embodiments a VHH isderived from a shark. In some embodiments, a provided binding agent isor comprises one or more “mini-antibodies” or “minibodies”, which aresFv polypeptide chains that include oligomerization domains at theirC-termini, separated from the sFv by a hinge region. In someembodiments, the hinge region comprises a self-associating alpha-helixor leucine zipper, which may or may not be further stabilized byadditional disulfide bonds.

In some embodiments, a binding agent is a scaffold protein such as, butis not limited to, protein A, lipoclins, ankryin consensus repeatdomain, thioredoxin, adnectin, anticalins, centyrin, avimer domains,ubiquitin, zinc finger DNA-binding proteins (ZEPs), or IgNARs. In someembodiments, a binding agent is a scaffold protein, in which thescaffold protein is engineered to display one or more CDRs. In someembodiments, a provided binding agent is or comprises a cystine-knotminiprotein. In some embodiments, a provided binding agent is orcomprises an avibody (diabody, tribody, tetrabody). In some embodiments,a provided binding agent is or comprises a Scorpion, wherein theScorpion structure comprises two binding moieties separated by animmunoglobulin Fc domain. In some embodiments, a provided binding agentis or comprises a peptidomimetic. In some embodiments, a providedbinding agent is or comprises a stapled peptide.

In some embodiments a binding agent comprises an agent that is capableof binding to a selected binding site. In some embodiments, a bindingagents is capable of binding to a selected binding site in ahemagglutinin (HA) polypeptide. In some specific embodiments, thebinding agents is capable of binding to a selected binding site in themembrane proximal epitope region (MPER) of a HA polypeptide. In somespecific embodiments, the binding agents is capable of binding to aselected binding site in the HA-1 (head) and/or HA-2 (stalk) domains ofan HA polypeptide. In some specific embodiments, the binding agents iscapable of binding to a selected binding site in the HA-1/HA-2 interfacemembrane proximal epitope region (MPER).

In some embodiments, a binding agent is an agent that is able toassociate with a binding target by interaction with one or more targetresidues. In some embodiments, such target residues are amino acids,saccharides, or combinations thereof. In some specific embodiments abinding agent is able to bind to a modified HA polypeptide such as, butnot limited to, N-linked glycans, sialylated glycans and/or combinationsthereof.

In some embodiments, a binding agent is an agent that is able to competewith an influenza virus for binding to an HA polypeptide, such thatbinding between the influenza virus and the HA polypeptide is reduced byat least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, atleast 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, atleast 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, atleast 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, atleast 17 fold, at least 18 fold, at least 19 fold, or at least 20 fold.In some embodiments, a binding agent is an agent that is able to competewith an influenza virus for binding to glycans on HA receptors such thatbinding between the influenza virus and the glycans on the HA receptoris reduced by at least 1.5 fold, at least 2 fold, at least 3 fold, atleast 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, atleast 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, atleast 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, atleast 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, orat least 20 fold.

In some embodiments, a binding agent is or comprises a nucleic acid,such as DNA or RNA. In some embodiments, a binding agent comprises oneor more oligonuclotides. In some embodiments, a binding agent is orcomprises one or more oligonuclotides comprising a secondary structuresuch as loop, hairpin, fold or combinations thereof. In someembodiments, a binding agent is or comprises one or more oligonuclotidescomprising a higher ordered (tertiary or quaternary) structure. In someembodiments a binding agent is a nucleic acid the forms a structuredesigned to mimic an epitope found within a hemagglutinin (HA)polypeptide. In some embodiments a binding agent is a nucleic acid theforms a structure designed to mimic a conserved epitope found within oneor more Influenza HA polypeptide subtypes. In some embodiments, abinding agent is or comprises an aptamer.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system (e.g., cell culture, organism, etc.). For instance, asubstance that, when administered to an organism, has a biologicaleffect on that organism, is considered to be biologically active. Inparticular embodiments, where a protein or polypeptide is biologicallyactive, a portion of that protein or polypeptide that shares at leastone biological activity of the protein or polypeptide is typicallyreferred to as a “biologically active” portion.

Broad spectrum: As used herein, the phrase “broad spectrum” refers toagents that bind a variety of HA polypeptides from different influenzavirus strains. In some embodiments, broad spectrum agents bind to aplurality of different HA polypeptides. Exemplary such HA polypeptidesinclude, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and/or H16 polypeptides, or combinations thereof. In someembodiments, provided agents are broad spectrum in that they bind to HApolypeptides from at least two different clades or clusters of virus. Insome embodiments provided agents are broad spectrum in that they bind toHA polypeptides from all known clades of virus. In some embodiments,provided agents are broad spectrum in that they bind to HA polypeptidesfrom group 1 and group 2 influenza viruses. In some embodiments, broadspectrum refers to agents that bind to some or all types of HApolypeptides that mediate infection of particular hosts, e.g., avian,camel, canine, cat, civet, equine, human, leopard, mink, mouse, seal,stone martin, swine, tiger, whale, etc.

Characteristic portion: As used herein, the term a “characteristicportion” of a substance, in the broadest sense, is one that shares somedegree of sequence or structural identity with respect to the wholesubstance. In certain embodiments, a characteristic portion shares atleast one functional characteristic with the intact substance. Forexample, a “characteristic portion” of a protein or polypeptide is onethat contains a continuous stretch of amino acids, or a collection ofcontinuous stretches of amino acids, that together are characteristic ofa protein or polypeptide. In some embodiments, each such continuousstretch generally contains at least 2, 5, 10, 15, 20, 50, or more aminoacids. In general, a characteristic portion of a substance (e.g., of aprotein, antibody, etc.) is one that, in addition to the sequence and/orstructural identity specified above, shares at least one functionalcharacteristic with the relevant intact substance. In some embodiments,a characteristic portion may be biologically active.

Characteristic sequence: A “characteristic sequence” is a sequence thatis found in all members of a family of polypeptides or nucleic acids,and therefore can be used by those of ordinary skill in the art todefine members of the family.

Combination Therapy: The term “combination therapy”, as used herein,refers to those situations in which two or more different agents areadministered in overlapping regimens so that the subject issimultaneously exposed to both agents.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic protein(e.g., antibody) for the patient to be treated. Each unit contains apredetermined quantity of active material calculated to produce thedesired therapeutic effect. It will be understood, however, that thetotal dosage of the composition will be decided by the attendingphysician within the scope of sound medical judgment.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regiment, which may involve one or more doses. Insome embodiments, a dosing regimen comprises a plurality of doses eachof which are separated from one another by a time period of the samelength; in some embodiments, a dosing regime comprises a plurality ofdoses and at least two different time periods separating individualdoses.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end formation); (3) translation of an RNA into a polypeptide orprotein; and/or (4) post-translational modification of a polypeptide orprotein.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Gene: As used herein, the term “gene” has its meaning as understood inthe art. It will be appreciated by those of ordinary skill in the artthat the term “gene” may include gene regulatory sequences (e.g.,promoters, enhancers, etc.) and/or intron sequences. It will further beappreciated that definitions of gene include references to nucleic acidsthat do not encode proteins but rather encode functional RNA moleculessuch as tRNAs, RNAi-inducing agents, etc. For the purpose of clarity wenote that, as used in the present application, the term “gene” generallyrefers to a portion of a nucleic acid that encodes a protein; the termmay optionally encompass regulatory sequences, as will be clear fromcontext to those of ordinary skill in the art. This definition is notintended to exclude application of the term “gene” to non-protein-codingexpression units but rather to clarify that, in most cases, the term asused in this document refers to a protein-coding nucleic acid.

Gene product or expression product: As used herein, the term “geneproduct” or “expression product” generally refers to an RNA transcribedfrom the gene (pre- and/or post-processing) or a polypeptide (pre-and/or post-modification) encoded by an RNA transcribed from the gene.

Hemagglutinin (HA) polypeptide: As used herein, the term “hemagglutininpolypeptide” (or “HA polypeptide”) refers to a polypeptide whose aminoacid sequence includes at least one characteristic sequence of HA. Awide variety of HA sequences from influenza isolates are known in theart; indeed, the National Center for Biotechnology Information (NCBI)maintains a database (www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) that,as of the filing of the present application included 9796 HA sequences.Those of ordinary skill in the art, referring to this database, canreadily identify sequences that are characteristic of HA polypeptidesgenerally, and/or of particular HA polypeptides (e.g., H1, H2, H3, H4,H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides;or of HA polypeptides that mediate infection of particular hosts, e.g.,avian, camel, canine, cat, civet, environment, equine, human, leopard,mink, mouse, seal, stone martin, swine, tiger, whale, etc.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% similar.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between nucleic acidmolecules (e.g., DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twonucleic acid sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or substantially 100% of the length of the reference sequence. Thenucleotides at corresponding nucleotide positions are then compared.When a position in the first sequence is occupied by the same nucleotideas the corresponding position in the second sequence, then the moleculesare identical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using the algorithm of Meyers and Miller(CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGNprogram (version 2.0) using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. The percent identity between twonucleotide sequences can, alternatively, be determined using the GAPprogram in the GCG software package using an NWSgapdna.CMP matrix.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.)

Mimotope: As used herein, the term “mimotope” refers to a macromoleculewhich mimics the structure of an epitope. In some embodiments, amimotope elicits an antibody response identical or similar to thatelicited by its corresponding epitope. In some embodiments, an antibodythat recognizes an epitope also recognizes a mimotope which mimics thatepitope. In some embodiments, a mimotope is a peptide. In someembodiments, a mimotope is a small molecule, carbohydrate, lipid, ornucleic acid. In some embodiments, mimotopes are peptide or non-peptidemimotopes of conserved influenza epitopes. In some embodiments, bymimicking the structure of a defined viral epitope, a mimotopeinterferes with the ability of influenza virus particles to bind to itsnatural binding partners, e.g., by binding to the natural bindingpartner itself.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into an oligonucleotide chain. In some embodiments, anucleic acid is a compound and/or substance that is or can beincorporated into an oligonucleotide chain via a phosphodiester linkage.In some embodiments, “nucleic acid” refers to individual nucleic acidresidues (e.g., nucleotides and/or nucleosides). In some embodiments,“nucleic acid” refers to an oligonucleotide chain comprising individualnucleic acid residues. As used herein, the terms “oligonucleotide” and“polynucleotide” can be used interchangeably. In some embodiments,“nucleic acid” encompasses RNA as well as single and/or double-strandedDNA and/or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,”and/or similar terms include nucleic acid analogs, i.e., analogs havingother than a phosphodiester backbone. For example, the so-called“peptide nucleic acids,” which are known in the art and have peptidebonds instead of phosphodiester bonds in the backbone, are consideredwithin the scope of the present invention. The term “nucleotide sequenceencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and/or encode the same amino acidsequence. Nucleotide sequences that encode proteins and/or RNA mayinclude introns. Nucleic acids can be purified from natural sources,produced using recombinant expression systems and optionally purified,chemically synthesized, etc. Where appropriate, e.g., in the case ofchemically synthesized molecules, nucleic acids can comprise nucleosideanalogs such as analogs having chemically modified bases or sugars,backbone modifications, etc. A nucleic acid sequence is presented in the5′ to 3′ direction unless otherwise indicated. The term “nucleic acidsegment” is used herein to refer to a nucleic acid sequence that is aportion of a longer nucleic acid sequence. In many embodiments, anucleic acid segment comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or moreresidues. In some embodiments, a nucleic acid is or comprises naturalnucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine);nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine); chemically modified bases;biologically modified bases (e.g., methylated bases); intercalatedbases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose); and/or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages). In someembodiments, the present invention is specifically directed to“unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotidesand residues, including nucleotides and/or nucleosides) that have notbeen chemically modified in order to facilitate or achieve delivery.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Polypeptide: As used herein, a “polypeptide”, generally speaking, is astring of at least two amino acids attached to one another by a peptidebond. In some embodiments, a polypeptide may include at least 3-5 aminoacids, each of which is attached to others by way of at least onepeptide bond. Those of ordinary skill in the art will appreciate thatpolypeptides sometimes include “non-natural” amino acids or otherentities that nonetheless are capable of integrating into a polypeptidechain, optionally.

Protein: As used herein, the term “protein” refers to a polypeptide(i.e., a string of at least two amino acids linked to one another bypeptide bonds). Proteins may include moieties other than amino acids(e.g., may be glycoproteins, proteoglycans, etc.) and/or may beotherwise processed or modified. Those of ordinary skill in the art willappreciate that a “protein” can be a complete polypeptide chain asproduced by a cell (with or without a signal sequence), or can be acharacteristic portion thereof. Those of ordinary skill will appreciatethat a protein can sometimes include more than one polypeptide chain,for example linked by one or more disulfide bonds or associated by othermeans. Polypeptides may contain l-amino acids, d-amino acids, or bothand may contain any of a variety of amino acid modifications or analogsknown in the art. Useful modifications include, e.g., terminalacetylation, amidation, methylation, etc. In some embodiments, proteinsmay comprise natural amino acids, non-natural amino acids, syntheticamino acids, and combinations thereof. The term “peptide” is generallyused to refer to a polypeptide having a length of less than about 100amino acids, less than about 50 amino acids, less than 20 amino acids,or less than 10 amino acids. In some embodiments, proteins areantibodies, antibody fragments, biologically active portions thereof,and/or characteristic portions thereof.

Reference Structural Element: As used herein, the term “referencestructural element” is an element of chemical structure against whichanother element is compared. In some embodiments, a reference structuralelement is or comprises an arrangement of atoms or moieties with respectto one another and/or in three dimensional space. In some suchembodiments, the relevant atoms or moieties are defined by chemicalidentity (e.g., a particular amino acid or chemical group or structure,etc) and/or by function (e.g., hydrogen-bond donor or acceptor, freeradical, etc). In some particular embodiments, where a referencestructural element is found in a reference polymer, the referencestructural element may be or comprise a sequence of monomers present inthe reference polymer. For example, in some such embodiments, areference structural element may be or comprise a particular amino acidsequence element in a polypeptide, a particular nucleotide sequenceelement in a polynucleotide, and or a particular glycan structure in apolysaccharide.

Small Molecule: In general, a “small molecule” is a molecule that isless than about 5 kilodaltons (kD) in size. In some embodiments, thesmall molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD.In some embodiments, the small molecule is less than about 800 daltons(D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, orabout 100 D. In some embodiments, a small molecule is less than about2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, lessthan about 800 g/mol, or less than about 500 g/mol. In some embodiments,small molecules are non-polymeric. In some embodiments, in accordancewith the present invention, small molecules are not proteins,polypeptides, oligopeptides, peptides, polynucleotides,oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantial homology: The phrase “substantial homology” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially homologous” ifthey contain homologous residues in corresponding positions. Homologousresidues may be identical residues. Alternatively, homologous residuesmay be non-identical residues will appropriately similar structuraland/or functional characteristics. For example, as is well known bythose of ordinary skill in the art, certain amino acids are typicallyclassified as “hydrophobic” or “hydrophilic” amino acids, and/or ashaving “polar” or “non-polar” side chains Substitution of one amino acidfor another of the same type may often be considered a “homologous”substitution.

As is well known in this art, amino acid or nucleic acid sequences maybe compared using any of a variety of algorithms, including thoseavailable in commercial computer programs such as BLASTN for nucleotidesequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acidsequences. Exemplary such programs are described in Altschul, et al.,Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990;Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al.,Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins,Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods andProtocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying homologous sequences, the programs mentionedabove typically provide an indication of the degree of homology. In someembodiments, two sequences are considered to be substantially homologousif at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues arehomologous over a relevant stretch of residues. In some embodiments, therelevant stretch is a complete sequence. In some embodiments, therelevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, 425, 450, 475, 500 or more residues.

Substantial identity: The phrase “substantial identity” is used hereinto refer to a comparison between amino acid or nucleic acid sequences.As will be appreciated by those of ordinary skill in the art, twosequences are generally considered to be “substantially identical” ifthey contain identical residues in corresponding positions. As is wellknown in this art, amino acid or nucleic acid sequences may be comparedusing any of a variety of algorithms, including those available incommercial computer programs such as BLASTN for nucleotide sequences andBLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplarysuch programs are described in Altschul, et al., Basic local alignmentsearch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al.,Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402,1997; Baxevanis et al., Bioinformatics: A Practical Guide to theAnalysis of Genes and Proteins, Wiley, 1998; and Misener, et al.,(eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifyingidentical sequences, the programs mentioned above typically provide anindication of the degree of identity. In some embodiments, two sequencesare considered to be substantially identical if at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more of their corresponding residues are identical over arelevant stretch of residues. In some embodiments, the relevant stretchis a complete sequence. In some embodiments, the relevant stretch is atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500 or more residues.

Suffering from: An individual who is “suffering from” a disease,disorder, or condition (e.g., influenza) has been diagnosed with and/orexhibits one or more symptoms of the disease, disorder, or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, or condition (e.g., influenza) is at risk for developing thedisease, disorder, or condition. In some embodiments, an individual whois susceptible to a disease, disorder, or condition does not display anysymptoms of the disease, disorder, or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, or condition hasnot been diagnosed with the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder, orcondition is an individual who has been exposed to conditions associatedwith development of the disease, disorder, or condition (e.g., theindividual has been exposed to influenza).

Symptoms are reduced: According to the present invention, “symptoms arereduced” when one or more symptoms of a particular disease, disorder orcondition is reduced in magnitude (e.g., intensity, severity, etc.) orfrequency. For purposes of clarity, a delay in the onset of a particularsymptom is considered one form of reducing the frequency of thatsymptom. It is not intended that the present invention be limited onlyto cases where the symptoms are eliminated. The present inventionspecifically contemplates treatment such that one or more symptomsis/are reduced (and the condition of the subject is thereby “improved”),albeit not completely eliminated.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that has a therapeutic effect and/or elicits a desiredbiological and/or pharmacological effect, when administered to asubject.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” refers to an amount of a therapeuticagent (e.g., protein, and specifically e.g., antibody) which isstatistically correlated with a particular therapeutic effect whenadministered to a population of subjects, at a reasonable benefit/riskratio applicable to any medical treatment. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). In particular, the“therapeutically effective amount” refers to an amount of a therapeuticagent effective to reduce the incidence and/or severity of and/or todelay onset of one or more features, symptoms, or characteristics of adisease, disorder, or condition. A therapeutically effective amount iscommonly administered in a dosing regimen that may comprise multipleunit doses. For any particular therapeutic protein, a therapeuticallyeffective amount (and/or an appropriate unit dose within an effectivedosing regimen) may vary, for example, depending on route ofadministration, on combination with other pharmaceutical agents. Also,the specific therapeutically effective amount (and/or unit dose) for anyparticular patient may depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific pharmaceutical agent employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and/orrate of excretion or metabolism of the specific fusion protein employed;the duration of the treatment; and like factors as is well known in themedical arts.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance (e.g., providedcompositions) that partially or completely alleviates, ameliorates,relives, inhibits, delays onset of, reduces severity of, and/or reducesincidence of one or more symptoms, features, and/or causes of aparticular disease, disorder, and/or condition (e.g., influenza). Suchtreatment may be of a subject who does not exhibit signs of the relevantdisease, disorder and/or condition and/or of a subject who exhibits onlyearly signs of the disease, disorder, and/or condition. Alternatively oradditionally, such treatment may be of a subject who exhibits one ormore established signs of the relevant disease, disorder and/orcondition. In some embodiments, treatment may be of a subject who hasbeen diagnosed as suffering from the relevant disease, disorder, and/orcondition. In some embodiments, treatment may be of a subject known tohave one or more susceptibility factors that are statisticallycorrelated with increased risk of development of the relevant disease,disorder, and/or condition.

Universal anti-influenza agent: As used herein, the term “universalanti-influenza agent” refers to an agent that has broad-spectrumneutralization across influenza virus strains, groups, clades, andclusters.

Unnatural amino acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue that is not one of the 20 naturally occurring amino acids.Refer to U.S. Pat. No. 7,045,337, U.S. Pat. No. 7,385,028, and U.S. Pat.No. 7,332,571, the entire disclosures of which are incorporated hereinby reference. As used herein, “unnatural amino acid” also encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and/or substitutions. Aminoacids, including carboxy- and/or amino-terminal amino acids in peptides,can be modified by PEGylation, methylation, amidation, acetylation,and/or substitution with other chemical groups that do not adverselyaffect the activity of the binding agent. Amino acids may participate ina disulfide bond. The term “amino acid” is used interchangeably with“amino acid residue,” and may refer to a free amino acid and/or to anamino acid residue of a peptide. It will be apparent from the context inwhich the term is used whether it refers to a free amino acid or aresidue of a peptide.

Vaccination: As used herein, the term “vaccination” refers to theadministration of a composition intended to generate an immune response,for example to a disease-causing agent. For the purposes of the presentinvention, vaccination can be administered before, during, and/or afterexposure to a disease-causing agent, and in certain embodiments, before,during, and/or shortly after exposure to the agent. In some embodiments,vaccination includes multiple administrations, appropriately spaced intime, of a vaccinating composition.

Vector: As used herein, “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it is associated.In some embodiment, vectors are capable of extra-chromosomal replicationand/or expression of nucleic acids to which they are linked in a hostcell such as a eukaryotic and/or prokaryotic cell. Vectors capable ofdirecting the expression of operatively linked genes are referred toherein as “expression vectors.”

DETAILED DESCRIPTION

As described herein, the present invention provides new influenzabinding agents that bind to multiple influenza strains. The presentinvention particularly provides certain antibodies that bind toinfluenza HA, along with binding agents that share particular structuraland/or functional characteristics of the provided antibodies.

Influenza Antigens

Hemagglutinin (HA) Polypeptides

Influenza viruses are RNA viruses which are characterized by a lipidmembrane envelope containing two glycoproteins, a hemagglutinin (HA)polypeptide and a neuraminidase (NA) polypeptide, embedded in themembrane of the virus particular. There are 16 known HA polypeptidesubtypes (H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and H16) and 9 NA polypeptide subtypes (N1, N2, N3, N4, N5, N6, N7,N8, and N9), and different influenza strains are named based on thenumber of the strain's HA polypeptide and NA polypeptide subtypes,wherein there are different combinations of one HA polypeptide subtypecombined with one NA polypeptide subtype (for example, H1N1, H1N2, H1N3,H1N4, H1N5, etc.).

Based on comparisons of amino acid sequence identity and of crystalstructures, the HA polypeptide subtypes have been divided into two maingroups and four smaller clades, which is further divided into fiveclusters. The different HA polypeptide subtypes do not necessarily sharestrong amino acid sequence identity, but the overall 3D structures ofthe different HA polypeptide subtypes are similar to one another, withseveral subtle differences that can be used for classification purposes.For example, the particular orientation of the membrane-distalsubdomains in relation to a central α-helix is one structuralcharacteristic commonly used to determine HA polypeptide subtype(Russell et al., Virology, 325:287, 2004).

HA, as it occurs in nature, is a trimer of three identical monomers,each synthesizes as a precursor that is proteolytically processed intotwo di-sulfide bonded polypeptide chains. Mature HA polypeptides arecomprised of two domains, (1) a core HA-1 domain known as the sialicacid-binding domain, and (2) the transmembrane stalk of HA, known asHA-2 domain. HA-1 contains the binding site for glycans and it isthought that HA-1 is responsible for mediating binding of HA to theHA-receptor. HA-2 is responsible for presenting the HA-1 domain.Typically, polar and non-polar interactions between the three long HAalpha-helices of the stem of HA monomers provide the main forces forstabilizing the HA trimer. It will be appreciated that HA polypeptidesin accordance with the present invention may contain amino acid residuesand/or sequences from any HA domain (e.g., core HA-1, transmembraneHA-2, and/or combinations thereof).

In some embodiments, an HA polypeptide in accordance with the presentinvention contains sequences that are conserved across more than oneinfluenza subtype. For example, analysis of HA sequences from allinfluenza subtypes showed a set of amino acids in the interface of theHA-1 (head) and HA-2 (stalk) domains that are well conserved andaccessible to prospective therapeutic molecules. Studies have alsoobserved the excellent broad spectrum conservation of the HA-1/HA-2interface membrane proximal epitope region (MPER) that includes thecanonical α-helix and residues in its vicinity (Ekiert et al., Science,324(5924):246, 2009; Sui et al., Nat Struct Mol Biol. 16(3):265, 2009).

HA Receptors

HA polypeptides as described herein interact with the surface of cellsby binding to a glycoprotein receptor, known as the HA receptor. Bindingof an HA polypeptide to an HA receptor is predominantly mediated byN-linked glycans on the HA receptors. Specifically, HA polypeptides onthe surface of flu virus particles recognizes sialylated glycans thatare associated with HA receptors on the surface of the cellular host.After recognition and binding, the host cell engulfs the viral cell andthe virus is able to replicate and produce many more virus particles tobe distributed to neighboring cells.

Natural HA (and in many embodiments, HA polypeptides) exist in the viralmembrane as a homotrimer of one of 16 subtypes, termed H1-H16. Onlythree of these subtypes (H1, H2, and H3) have thus far become adaptedfor human infection. One reported characteristic of HA polypeptides thathave adapted to infect humans (e.g., of HA polypeptides from thepandemic H1N1 (1918) and H3N2 (1967-68) influenza subtypes) is theirability to preferentially bind to α2,6 sialylated glycans in comparisonwith their avian progenitors that preferentially bind to α2,3 sialylatedglycans (Skehel & Wiley, Annu Rev Biochem, 69:531, 2000; Rogers, &Paulson, Virology, 127:361, 1983; Rogers et al., Nature, 304:76, 1983;Sauter et al., Biochemistry, 31:9609, 1992; Connor et al., Virology,205:17, 1994; Tumpey et al., Science, 310:77, 2005).

Without wishing to be bound by any particular theory, it has beenproposed that the ability to infect human hosts correlates less withbinding to glycans of a particular linkage, and more with binding toglycans of a particular topology. We have specifically demonstrated thatHA polypeptides that mediate infection of humans bind to umbrellatopology glycans, often showing preference for umbrella topology glycansover cone topology glycans (even though cone-topology glycans may beα2,6 sialylated glycans) (See, for example, U.S. Ser. No. 12/348,266filed Jan. 2, 2009, U.S. Ser. No. 12/301,126, filed Nov. 17, 2008, U.S.Ser. No. 61/018,783, filed Jan. 3, 2008, U.S. Ser. No. 11/969,040, filedJan. 3, 2008, U.S. Ser. No. 11/893,171, filed Aug. 14, 2007, U.S. Ser.No. 60/837,868, filed on Aug. 14, 2006, U.S. Ser. No. 60/837,869, filedon August 14, and to PCT application PCT/US07/18160, filed Aug. 14,2007, each of which is incorporated herein by reference).

Several crystal structures of HA polypeptides from H1 (human and swine),H3 (avian) and H5 (avian) subtypes bound to sialylated oligosaccharides(of both α2,3 and α2,6 linkages) are available and provide molecularinsights into the specific amino acids that are involved in distinctinteractions of the HA polypeptides with these glycans (Eisen et al.,Virology, 232:19, 1997; Ha et al., Proc Natl Acad Sci USA, 98:11181,2001; Ha et al., Virology, 309:209, 2003; Gamblin et al., Science,303:1838, 2004; Stevens et al., Science, 303:1866, 2004; Russell et al.,Glycoconj J 23:85, 2006; Stevens et al., Science, 312:404, 2006). Somecrystal structures of exemplary HA-glycan interactions have beenidentified and are presented in Table 1 below.

TABLE 1 Crystal Structures of HA-Glycan Complexes Abbreviation (PDB ID)Virus Strain Glycan (with assigned coordinates) ADkALB76_H1_26 (2WRH)A/duck/Alberta/76 (H1N1) Neu5Ac ASI30_H1_23 (1RV0) A/Swine/Iowa/30(H1N1) Neu5Ac ASI30_H1_26 (1RVT) A/Swine/Iowa/30 (H1N1)Neu5Acα6Galβ4GlcNAcβ3Galβ4Glc ASC18_H1_26 (2WRG) A/South Carolina/1/18(H1N1) Neu5Acα6Galβ4GlcNAcβ3Gal APR34_H1_23 (1RVX) A/Puerto Rico/8/34(H1N1) Neu5Acα3Galβ4GlcNAc APR34_H1_26 (1RVZ) A/Puerto Rico/8/34 (H1N1)Neu5Acα6Galβ4GlcNAc ACkNY91_H2_23 (2WR2) A/chicken/NY/29878/91 (H2N2)Neu5Acα3Galβ3GlcNAc AckNY91_H2_26 (2WR1) A/chicken/NY/29878/91 (H2N2)Neu5Acα6Galβ4GlcNAc AdkON77_H2_23 (2WR3) A/duck/Ontario/77 (H2N2)Neu5Acα3Galβ4GlcNAc AdkON77_H2_26 (2WR4) A/duck/Ontario/77 (H2N2)Neu5Acα6Galβ4GlcNAc AckPD84_H2_26 (2WRF) A/chicken/Potsdam/475/84 (H2N2)Neu5Acα6Gal ASING57_H2_23 (2WRB) A/Singapore/1/57 (H2N2) Neu5AcASING57_H2_26 (2WR7) A/Singapore/1/57 (H2N2) Neu5Acα6Galβ4GlcNAcβ3GalAJAP57_H2_26(2WRE) A/Japan/305/57 (H2N2) Neu5Acα6Gal ADU63_H3_23 (1MQM)A/Duck/Ukraine/1/63 (H3N8) Neu5Acα3Gal ADU63_H3_26 (1MQN)A/Duck/Ukraine/1/63 (H3N8) Neu5Acα6Gal AAI68_H3_23 (1HGG) A/Aichi/2/68(H3N2) Neu5Acα3Galβ4Glc ADS97_H5_23 (1JSN) A/Duck/Singapore/3/97 (H5N3)Neu5Acα3Galβ3GlcNAc ADS97_H5_26(1JSO) A/Duck/Singapore/3/97 (H5N3)Neu5Ac Viet1203_04_H5 (2FK0) A/Vietnam/1203/2004 (H5N1) Viet1194_04_H5(2IBX) A/Vietnam/1194/2004 (H5N1) ASI30_H1_23 (1RV0) A/Swine/Iowa/30(H1N1) Neu5Ac ASI30_H1_26 (1RVT) A/Swine/Iowa/30 (H1N1)Neu5Acα6Galβ4GlcNAcβ3Galβ4Glc APR34_H1_23 (1RVX) A/Puerto Rico/8/34(H1N1) Neu5Acα3Galβ4GlcNAc APR34_H1_26 (1RVZ) A/Puerto Rico/8/34 (H1N1)Neu5Acα6Galβ4GlcNAc ADU63_H3_23 (1MQM) A/Duck/Ukraine/1/63 (H3N8)Neu5Acα3Gal ADU63_H3_26 (1MQN) A/Duck/Ukraine/1/63 (H3N8) Neu5Acα6GalAAI68_H3_23 (1HGG) A/Aichi/2/68 (H3N2) Neu5Acα3Galβ4Glc ADS97_H5_23(1JSN) A/Duck/Singapore/3/97 (H5N3) Neu5Acα3Galβ3GlcNAcADS97_H5_26(1JSO) A/Duck/Singapore/3/97 (H5N3) Neu5Ac Viet04_H5 (2FK0)A/Vietnam/1203/2004 (H5N1)HA-α2-6 sialylated glycan complexes were generated by superimposition ofthe CA trace of the HA1 subunit of ADU63_H3 and ADS97_H5 and Viet04_H5on ASI30_H1_26 and APR34_H1_26 (H1). Although the structural complexesof the human A/Aichi/2/68 (H3N2) with α2-6 sialylated glycans arepublished (Eisen et al., 1997, Virology, 232:19; incorporated herein byreference), their coordinates were not available in the Protein DataBank. The SARF2 program was used to obtain the structural alignment ofthe different HA1 subunits for superimposition.

For example, crystal structures of H5 (A/duck/Singapore/3/97) alone orbound to an α2,3 or an α2,6 sialylated oligosaccharide identifiescertain amino acids that interact directly with bound glycans, and alsoamino acids that are one or more degree of separation removed (Stevenset al., Proc Natl Acad Sci USA 98:11181, 2001). In some cases,conformation of these residues is different in bound versus unboundstates. For instance, Glu190, Lys193 and Gln226 all participate indirect-binding interactions and have different conformations in thebound versus the unbound state. The conformation of Asn186, which isproximal to Glu190, is also significantly different in the bound versusthe unbound state.

Without wishing to be bound by any particular theory, it is thought thatthe HA receptors are modified by either α2,3 or α2,6 sialylated glycansnear the receptor's HA polypeptide-binding site, and the type of linkageof the receptor-bound glycan can affect the conformation of thereceptor's HA polypeptide-binding site, thus affecting the receptor'sspecificity for different HA polypeptides. For example, the glycanbinding pocket of avian HA receptor is narrow. Without wishing to bebound by any particular theory, it has been proposed that this pocketbinds to the trans conformation of α2,3 sialylated glycans, and/or tocone-topology glycans, whether α2,3 or α2,6 linked.

HA receptors in avian tissues, and also in human deep lung andgastrointestinal (GI) tract tissues are characterized by α2,3 sialylatedglycan linkages, and furthermore are characterized by glycans, includingα2,3 sialylated and/or α2,6 sialylated glycans, which predominantlyadopt cone topologies. HA receptors having such cone-topology glycansmay be referred to herein as CTHArs.

By contrast, human HA receptors in the bronchus and trachea of the upperrespiratory tract are modified by α2,6 sialylated glycans. Unlike theα2,3 motif, the α2,6 motif has an additional degree of conformationalfreedom due to the C6-05 bond (Russell et al., Glycoconj J 23:85, 2006).HA polypeptides that bind to such α2,6 sialylated glycans have a moreopen binding pocket to accommodate the diversity of structures arisingfrom this conformational freedom. Moreover, according to the presentinvention, HA polypeptides may need to bind to glycans (e.g., α2,6sialylated glycans) in an umbrella topology, and particularly may needto bind to such umbrella topology glycans with strong affinity and/orspecificity, in order to effectively mediate infection of human upperrespiratory tract tissues. HA receptors having umbrella-topology glycansmay be referred to herein as UTHArs.

As a result of these spatially restricted glycosylation profiles, humansare not usually infected by viruses containing many wild type avian HApolypeptides (e.g., avian H5). Specifically, because the portions of thehuman respiratory tract that are most likely to encounter virus (i.e.,the trachea and bronchi) lack receptors with cone glycans (e.g., α2,3sialylated glycans, and/or short glycans) and wild type avian HApolypeptides typically bind primarily or exclusively to receptorsassociated with cone glycans (e.g., α2,3 sialylated glycans, and/orshort glycans), humans rarely become infected with avian viruses. Onlywhen in sufficiently close contact with virus that it can access thedeep lung and/or gastrointestinal tract receptors having umbrellaglycans (e.g., long α2,6 sialylated glycans) do humans become infected.

Symptoms and Effects of Influenza Infection

Influenza infection or “flu” is a viral infection predominantly of thenose, throat and bronchial tubes of the human body. As described above,given the presence of several different influenza strains and subtypes,severity of the symptoms associate with influenza infection can varydepending on the type of infection. Symptoms can also become lifethreatening in those individuals with chronic underlying illnesses (suchas cancer, emphysema or diabetes) or those who are immunocompromised.While the severity of the symptoms may vary, there are several hallmarksymptoms of an influenza infection such as, but not limited to,inflammation, fever, nausea, weight loss, loss of appetite, rapidbreathing, increase heart rate, high blood pressure, body aches, musclepain, eye pain, fatigue, malaise, dry cough, runny nose, and/or sorethroat. As a result, in some embodiments, the manifestation of symptomswithin a patient can be used as a prognostic or diagnostic to determinethe presence of an influenza infection. In some embodiments, theseverity and/or change of symptoms may be used to determined the dosingregiment for an influenza treatment, such as administration of a bindingagent. In some embodiments, the onset, severity and/or change ofsymptoms displayed by the patient, may be used to indicate the need forprophylactic treatment of a patient with a binding agent. In someembodiments, the severity, change and/or amelioration of symptoms may beused to evaluate a patient's response to a specific type or method ofinfluenza treatment.

Antibodies

As described herein, the present invention provides antibodies (e.g.,monoclonal antibodies, human antibodies, humanized antibodies, etc.)that bind to HA polypeptides, and in some embodiments bind to multipleinfluenza strains. In some embodiments, such antibodies are useful inthe prophylaxis, treatment, diagnosis, and/or study of influenza.

As used herein, an antibody refers to a polypeptide having structuralcharacteristics of an immunoglobulin, as those are understood in theart. In many embodiments, an antibody is a polypeptide whose amino acidsequence includes one or more structural elements recognized by thoseskilled in the are as a complementarity determining region (CDR). Insome embodiments, an antibody is a polypeptide whose amino acid sequenceincludes structural elements recognized by those skilled in the art asan immunoglobulin variable domain. In some embodiments, an antibody is“full length” in that in contains two heavy chains and two light chains,optionally associated by disulfide bonds as occurs withnaturally-produced antibodies. In some embodiments, an antibody is afragment of a full-length antibody in that it contains some, but not allof the sequences found in a full-length antibody. For example, in someembodiments, Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fd fragments. Insome embodiments, an antibody may comprise a single chain antibodyfragment. In some embodiments, an antibody may comprise multiple chainswhich are linked together, for example by disulfide linkages. In someembodiments, an antibody may comprise a multimolecular complex. In someembodiments, an antibody may comprises at least about 50 amino acids andmore typically comprises at least about 200 amino acids.

In some embodiments, an antibody is a member an antibody class selectedfrom the group consisting of IgG, IgM, IgA, IgD, and IgE. In someembodiments, an antibody is produced by a cell. In some embodiments, anantibody is produced using a recombinant cell culture system. In someembodiments, an antibody is produced by chemical synthesis. In someembodiments, an antibody is a monoclonal antibody. In some embodiments,an antibody is a polyclonal antibody. In some embodiments, an antibodyis produced by chemical synthesis. In some embodiments, an antibody isderived from a mammal. In some embodiments, an antibody is produced byan animal such as, but not limited to, mouse, rat, horse, pig, or goat.In some embodiments, an antibody is a derived from a human. In someembodiments an antibody is a chimeric antibody, for example, from mouse,rat, horse, pig, or other species, bearing human constant and/orvariable regions domains. In some embodiments, an antibody is ahumanized antibody. As used herein, the term “humanized” antibody refersto an immunoglobulin comprising a human framework region and one or moreCDR's from a non-human (e.g., mouse or rat) immunoglobulin. Thenon-human immunoglobulin providing the CDR's is called the “donor” andthe human immunoglobulin providing the framework is called the“acceptor”. A “humanized antibody” is an antibody comprising a humanizedlight chain and a humanized heavy chain immunoglobulin.

Antibody Modification

In some embodiments, one or more sequences in a provided antibody hasbeen engineered (e.g., by affinity maturation or other optimizationapproach) to improve one or more characteristics or activities (e.g., toincrease stability, decrease aggregation, decrease immunogenicity, etc)as is known in the art. In some embodiments, an antibody is modified byPEGylation, methylation, sialylation, amination or sulfation. In someembodiments, an antibody is conjugated to an amphiphilic core/shell toproduce a polymeric micelle. In some embodiments, an antibody isconjugated to a hyperbranched macromolecule (i.e. dendrimer). In someembodiments, an antibody is conjugated to a natural polymer selectedfrom the group consisting of albumin, chitosan, heparin, paclitaxel,poly-(L-glutamate), N-(2-hydroxypropyl)methacrylamide (HPMA),poly-(L-lactide) (PLA), poly(amidoamine) (PAMAM), folate and/orcombinations thereof. In some embodiments, an antibody comprises one ormore long unstructured tails of hydrophilic amino acids (rPEG). In someembodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology may exhibit improved longevity, specificity and sensitivity(U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987).

Provided Anti-HA Antibodies

The present disclosure provides certain antibodies that bind toinfluenza HA. The present invention particularly provides certainantibodies which are characterized by binding to a particular HA epitopeand/or to a HA from one or more influenza groups. In some embodiments,such antibodies are characterized by binding to a particular HA epitopeand/or to an HA from a group 1 virus. In some embodiments, suchantibodies are characterized by binding to a particular HA epitopeand/or to an HA from a group 2 virus. In some embodiments, suchantibodies are characterized by binding to a particular HA epitopeand/or to an HA from a group 1 virus and a group 2 virus. In someembodiments, such antibodies bind to a HA polypeptides of subtype H1,H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and/orH16. Specifically, in some embodiments, such antibodies bind to HApolypeptides that have sequence elements characteristic of one or moreof H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 andH16 HA polypeptides. In some embodiments, such antibodies bind to one ormore of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15and H16 HA polypeptides with an affinity that is at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or more of its affinity for one or more of a different H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 HApolypeptides. In some embodiments such antibodies show bindingaffinities for different HA polypeptides (e.g., HA polypeptides fromdifferent groups, clades, or clusters and/or from different strains)that are within 5 fold binding affinity of one another. In someembodiments such antibodies show binding affinities for different HApolypeptides that are within 2 fold of one another. In some embodimentssuch antibodies show binding affinities for different HA polypeptides(e.g., HA polypeptides from different groups, clades, or clusters and/orfrom different strains) that are within 150 fold (e.g., within 100 fold,within 50 fold, within 25 fold, within 10 fold, or within 5 fold)binding affinity of one another.

In some embodiments, such antibodies bind to at least two of H1, H3, H5,H7, and/or H9 HA polypeptides. In some embodiments, such antibodies bindto at least three, four or five of the H1, H3, H5, H7, and/or H9 HApolypeptides.

In some embodiments, such antibodies bind to HA polypeptides of at leastone of subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15, and/or H16, and do not bind to at least one HA polypeptide ofsubtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15, and/or H16. In some embodiments, such antibodies bind to HApolypeptides of subtype H1. In some embodiments, such antibodies bind toHA polypeptides of subtype H1 with an affinity at least 100%, at least125%, at least 150%, at least 200% or more of that with which it bindsto HA polypeptides of at least one subtype H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15, and/or H16. In some embodiments, suchantibodies bind to HA polypeptides of subtype H3. In some embodiments,such antibodies bind to HA polypeptides of subtype H3 with an affinityat least 100%, at least 125%, at least 150%, at least 200% or more ofthat with which it binds to HA polypeptides of at least one subtype H1,H2, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and/or H16.

In some embodiments, such antibodies show a neutralization IC₅₀ (ug/ml)within a range as described and/or exemplified herein. In someembodiments, such provided antibodies show a neutralization IC₅₀ (ug/ml)whose lower bound is about 0.1 ug/ml and upper bound is about 10 ug/ml.In some embodiments, such provided antibodies show a neutralization IC₅₀(ug/ml) whose lower bound is selected from the group consisting of 0.05,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or moreug/ml, and whose upper bound is higher than the lower bound and isselected from the group consisting of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0 or more ug/ml.

In some embodiments, such provided antibodies show binding to influenzaHA (e.g., group 1 and/or group 2 subtypes) with a K_(D) (nM) less than2000 nM, less than 1500 nM, less than 1000 nM, less than 500 nM, lessthan 250 nM, less than 225 nM, less than 200 nM, less than 175 nM, lessthan 150 nM, less than 125 nM, less than 100 nM, less than 75 nM, orless than 50 nM.

In some embodiments, such provided antibodies show binding to influenzaHA with a K_(a) (M⁻¹s⁻¹) whose lower bound is about 0.01×10⁵ M⁻¹s⁻¹ andupper bound is about 1.0×10⁶ M⁻¹s⁻¹. such provided antibodies showbinding to influenza HA with a K_(a) (M⁻¹s⁻¹) whose lower bound isselected from the group consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵,0.04×10⁵, 0.08×10⁵, 0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵,1.0×10⁵, 1.2×10⁵, 1.4×10⁵, 1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more M⁻¹s⁻¹,and whose upper bound is higher than the lower bound and is selectedfrom the group consisting of 1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵,3.0×10⁵, 3.5×10⁵, 4.5×10⁵, 5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵,7.5×10⁵, 8.0×10⁵, 8.5×10⁵, 9.0×10⁵, 9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶,1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or moreM⁻¹s⁻¹.

In some embodiments, such provided antibodies show binding to influenzaHA with a K_(d) (s⁻¹) whose lower bound is about 0.01×10⁵ s⁻¹ and upperbound is about 1.0×10⁶ s⁻¹. such provided antibodies show binding toinfluenza HA with a K_(a) (s⁻¹) whose lower bound is selected from thegroup consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵, 0.04×10⁵, 0.08×10⁵,0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵, 1.0×10⁵, 1.2×10⁵, 1.4×10⁵,1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more s⁻¹, and whose upper bound is higherthan the lower bound and is selected from the group consisting of1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵, 3.0×10⁵, 3.5×10⁵, 4.5×10⁵, 5.0×10⁵,5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵, 7.5×10⁵, 8.0×10⁵, 8.5×10⁵, 9.0×10⁵,9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or more s⁻¹.

In some embodiments, such provided antibodies are characterized by aspecific structural feature. In some embodiments, such structuralfeatures of such certain provided antibodies include one or more CDRs orone or more FRs at least 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98 or 99% identical in sequence to a correspondingCDR or FR from Tables 2 and 3 (SEQ ID NO: 1-60). In some embodiments,such structural features of such certain provided antibodies include oneor more CDRs or one or more FRs comprising at least 65, 70, 75, 80, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% homology to acorresponding CDR or FR from Tables 2 and 3 (SEQ ID NO: 1-60). In someembodiments, such structural features of such certain providedantibodies include one or more CDRs and/or one or more FRs that isidentical in sequence to a corresponding CDR or FR from Tables 2 and 3(SEQ ID NO: 1-60). In some embodiments, such structural features of suchcertain provided antibodies include CDRs and FRs that are identical insequence to those set forth in Tables 2 and 3 (SEQ ID NO: 1-60).

TABLE 2Exemplary Amino Acid Sequence of VH Chain (CDR Sequences in bold)Exemplary Amino VH Acid Sequence (CDR Framework Sequences in bold) CDR 1CDR 2 CDR 3  1 EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDSELRSLLYFEWLSQGYFNP GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 21) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS ELRSLLYFEWLSQGYFNPWGAGTTLTVSSASTK(SEQ ID NO: 1)  2 EVQLLESGGGVVQPGRSLKLSCAAS GFTFSSY SYDGSNDGKLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQAPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 22) ISYDGSNKYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDG KLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 2)  3 EVQLLESGGGVVQPGRSLKLSCAAS GFTFSSY SYDGSNDGKLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQAPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 22) VSYDGSNKYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDG KLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 3)  4 EVQLLESGGGVVQPGRSLKLSCAAS GFTFSSY SYDGSNDGKLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQAPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 22) VSYDGSNKYYAPKFEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDG KLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 4)  5 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSNDGKLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 22) VSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDG KLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 5)  6 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSNDSQLRSLVYFEWLSSGLLDY GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 23) VSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKDS QLRSLVYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 6)  7 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSNDSQLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 24) VSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS QLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 7)  8 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSNDSKLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 22) VSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS KLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 8)  9 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSNDTQLRTIVYFEWLSQGFYDI GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 25) VSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDT QLRTIVYFEWLSQGFYDIWGQGAMVTVSSASTK (SEQ ID NO: 9) 10 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSNDSQLRTIVYFEWLSQGYFDP GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 20) (SEQ ID NO: 26) VSYDGSNKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS QLRTIVYFEWLSQGYFDPWGQGAMVTVSSASTK (SEQ ID NO: 10) 11 EVQLLESGGGLVKPGQSLKLSCAAS GFTFSSY SYDGSYDSQLRSLLYFEWLSSGLLDY GFTFSSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 18)(SEQ ID NO: 19) (SEQ ID NO: 27) VSYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS QLRSLLYFEWLSSGLLDYWGQGAMVTVSSASTK (SEQ ID NO: 11) 12 EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDSQLRSLIYFEWLSNGYFDI GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 28) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKDS QLRSLIYFEWLSNGYFDIWGAGTTLTVSSASTK (SEQ ID NO: 12) 13 EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDSQLRSLLYFEWLSNGFYDI GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 29) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS QLRSLLYFEWLSNGFYDIWGAGTTLTVSSASTK (SEQ ID NO: 13) 14 EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDSNLRTIVYFEWLSSGLLDY GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 30) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS NLRTIVYFEWLSSGLLDYWGAGTTLTVSSASTK (SEQ ID NO: 14) 15 EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDSQLRTIVYFEWLSQGYFDP GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 31) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS QLRTIVYFEWLSQGYFDPWGAGTTLTVSSASTK (SEQ ID NO: 15) 16 EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDTQLRTIVYFEWLSQGFYDI GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 32) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDT QLRTIVYFEWLSQGFYDIWGAGTTLTVSSASTK (SEQ ID NO: 16)

TABLE 3Exemplary Amino Acid Sequence of VL Chain (CDR Sequences in bold)Exemplary Amino VL Acid Sequence (CDR Framework Sequences in bold) CDR 1CDR 2 CDR 3  1 EIVMTQSPDSLAVSLGERATINC KSSQSVTYNYKNYLA WASTRES QQYYRTPPTKSSQSVTYNYKNYLAWYQQKPGQ (SEQ ID NO: 44) (SEQ ID NO: 55) (SEQ ID NO: 59)PPKLLIYWASTRESGVPDRFSGS GSGTDFTLTISSLQAEDVAVYYC QQYYRTPPTFGGGTKLDIKGS(SEQ ID NO: 33)  2 DIQMTQSPSSLSASVGDRVTITC RASQDVNTAVA SASFLYS QQHYTTPPTRASQDVNTAVAWYQQKPGKAPKL (SEQ ID NO: 45) (SEQ ID NO: 56) (SEQ ID NO: 60)LIYSASFLYSGVPSRFSGSRSGT DFTLTISSLQPEDFATYYCQQHY TTPPTFGQGTKVEIKGS(SEQ ID NO: 34)  3 DIQMTQSPSSLSASVGDRVTITC RASQDIPRSISGYVA WGSYLYSQQHYTTPPT RASQDIPRSISGYVAWYQQKPGK (SEQ ID NO: 46) (SEQ ID NO: 57)(SEQ ID NO: 60) APKLLIYWGSYLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGS (SEQ ID NO: 35)  4 DIQMTQSPSSLSASVGDRVTITCRASQDIPFSYKGYVA WGSYLES QQHYTTPPT RASQDIPFSYKGYVAWYQQKPGK(SEQ ID NO: 47) (SEQ ID NO: 57) (SEQ ID NO: 60) APKLLIYWGSYLESGVPSRFSGSExemplary VL GSGTDFTLTISSLQPEDFATYYC Chain QQHYTTPPTFGQGTKVEIKGS(SEQ ID NO: 36)  5 DIQMTQSPSSLSASVGDRVTITC RASQSITFDYKNYVA WGSYLEQQHYTTPPT RASQSITFDYKNYVAWYQQKPGK (SEQ ID NO: 48) (SEQ ID NO: 58)(SEQ ID NO: 60) APKLLIYWGSYLEGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGS (SEQ ID NO: 37)  6 DIQMTQSPSSLSASVGDRVTITCRASQSITFNYKNYVA WGSYLES QQHYTTPPT RASQSITFNYKNYVAWYQQKPGK(SEQ ID NO: 49) (SEQ ID NO: 57) (SEQ ID NO: 60) APKLLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYTTPPTFGQGTKVEIKGS (SEQ ID NO: 38)  7DIQMTQSPSSLSASVGDRVTITC RASQSITFSYKNYVA WGSYLES QQHYTTPPTRASQSITFSYKNYVAWYQQKPGK (SEQ ID NO: 50) (SEQ ID NO: 57) (SEQ ID NO: 60)APKLLIYWGSYLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYC QQHYTTPPTFGQGTKVEIKGS(SEQ ID NO: 39)  8 DIQMTQSPSSLSASVGDRVTITC RASQDIPFSYKGYVA WGSYLESQQHYTTPPT RASQDIPFSYKGYVAWYQQKPGK (SEQ ID NO: 51) (SEQ ID NO: 57)(SEQ ID NO: 60) APKVLIYWGSYLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGS (SEQ ID NO: 40)  9 DIQMTQSPSSLSASVGDRVTITCRASQSITFDYKNYVA WGSYLES QQHYTTPPT RASQSITFDYKNYVAWYQQKPGK(SEQ ID NO: 52) (SEQ ID NO: 57) (SEQ ID NO: 60) APKVLIYWGSYLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYTTPPTFGQGTKVEIKGS (SEQ ID NO: 41) 10DIQMTQSPSSLSASVGDRVTITC RASQSITFNYKNYVA WGSYLES QQHYTTPPTRASQSITFNYKNYVAWYQQKPGK (SEQ ID NO: 53) (SEQ ID NO: 57) (SEQ ID NO: 60)APKVLIYWGSYLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYC QQHYTTPPTFGQGTKVEIKGS(S(SEQ ID NO: 42) 11 DIQMTQSPSSLSASVGDRVTITC RASQSITFSYKNYVA WGSYLESQQHYTTPPT RASQSITFSYKNYVAWYQQKPGK (SEQ ID NO: 54) (SEQ ID NO: 57)(SEQ ID NO: 60) APKVLIYWGSYLESGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGS (SEQ ID NO: 43)

In some embodiments, such structural features include CDR and FRsequence elements, each of which is identical to a reference CDR or FRsequence element set forth in Table 2 and/or Table 3 (SEQ ID NOs:1-60)except that it includes one or more amino acid substitutions withrespect to that reference sequence element, where the included CDR andFR sequence elements together contain no more than 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in CDR and FRsequences, as compared with the corresponding CDR and FR referencesequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). In someembodiments, such structural features include CDR and FR sequenceelements together contain no more than 18 substitutions as compared withthe corresponding CDR and FR reference sequence elements from Tables 2and 3 (SEQ ID NO: 1-60). In some embodiments, such structural featuresinclude CDR and FR sequence elements together contain no more than 15substitutions as compared with the corresponding CDR and FR referencesequence elements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features include a FR sequenceelement, which is identical to a reference FR sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included FR sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in FR sequence, as compared with the corresponding FRreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features include a CDR sequenceelement, which is identical to a reference CDR sequence element setforth in Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that itincludes one or more amino acid substitutions with respect to thatreference sequence element, where the included CDR sequence elementcontains no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 substitutions in CDR sequence, as compared with thecorresponding CDR reference sequence elements from Tables 2 and 3 (SEQID NO: 1-60).

In some embodiments, such structural features include a VH sequenceelement, which is identical to a reference VH sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included VH sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in CDR sequence, as compared with the corresponding VHreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). Insome embodiments, such structural features include a structural elementcorresponding to any one of VH-1, VH-2, VH-3, VH-4, VH-5, VH-6, VH-7,VH-8, VH-9, VH-10, VH-11, VH-12, VH-13, VH-14, VH-15, VH-16 or fragmentthereof, as compared with the corresponding VH reference sequenceelements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features include a VL sequenceelement, which is identical to a reference VL sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included VL sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in CDR sequence, as compared with the corresponding VHreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). Insome embodiments, such structural features include a structural elementcorresponding to any one of VL-1, VL-2, VL-3, VL-4, VL-5, VL-6, VL-7,VL-8, VL-9, VL-10, VL-11, or fragment thereof, as compared with thecorresponding VL reference sequence elements from Tables 2 and 3 (SEQ IDNO: 1-60).

In some embodiments, such structural features include a structuralelement corresponding to any one of VH-1, VH-2, VH-3, VH-4, VH-5, VH-6,VH-7, VH-8, VH-9, VH-10, VH-11, VH-12, VH-13, VH-14, VH-15, VH-16 orfragment thereof combined with any one of VL-1, VL-2, VL-3, VL-4, VL-5,VL-6, VL-7, VL-8, VL-9, VL-10, VL-11, or fragment thereof. In someembodiments, VH-1 (SEQ ID NO:1) is combined with VL-1 (SEQ ID NO:33).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR1sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 1 sequence element that has twoor more amino acid substitutions as compared to a reference CDR1sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 1 sequence element that has oneor more amino acid substitutions as compared to reference CDR1 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has at least two aminoacid substitutions as compared to reference CDR1 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR1 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR1 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR2sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 2 sequence element that has twoor more amino acid substitutions as compared to a reference CDR2sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 2 sequence element that has oneor more amino acid substitutions as compared to reference CDR2 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has at least two aminoacid substitutions as compared to reference CDR2 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR2 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR2 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR3sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 3 sequence element that has twoor more amino acid substitutions as compared to a reference CDR3sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 3 sequence element that has oneor more amino acid substitutions as compared to reference CDR3 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has at least two aminoacid substitutions as compared to reference CDR3 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR3 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR3 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a VH frameworkregion sequence element that shows more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 99% percent identity with a reference VH framework regionsequence element from Table 2 (SEQ ID NOs:1-16).

In some embodiments, such structural features include a VL frameworkregion sequence element that shows more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 99% percent identity with a reference VL framework regionsequence element from Table 3 (SEQ ID NOs:33-43).

Binding Agents

Structures and Features

The present disclosure provides influenza binding agents. The presentinvention particularly provides binding agents that include suchstructural features of such certain provided antibodies such that theprovided binding agent shares with the certain provided influenzaantibodies the functional attribute that it binds to a particular HAepitope and/or to a HA from a specific influenza group. In someembodiments, such binding agent is characterized by a functionalattribute of binding to a particular HA epitope and/or to a HA from agroup 1 virus. In some embodiments, such binding agent is characterizedby a functional attribute of binding to a particular HA epitope and/orto a HA from a group 2 virus. In some embodiments, such binding agent ischaracterized by a functional attribute of binding to a particular HAepitope and/or to a HA from a group 1 virus and a group 2 virus. In someembodiments, such binding agent is characterized by a functionalattribute of binding to an HA polypeptides of subtype H1, H2, H3, H4,H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and/or H16.Specifically, in some embodiments, such binding agent is characterizedby a functional attribute of binding to HA polypeptides that havesequence elements characteristic of one or more of H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and H16 HA polypeptides. Insome embodiments, such binding agent is characterized by a functionalattribute of binding to one or more of H1, H2, H3, H4, H5, H6, H7, H8,H9, H10, H11, H12, H13, H14, H15 and H16 HA polypeptides with anaffinity that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of its affinity forone or more of a different H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,H12, H13, H14, H15 and H16 HA polypeptides.

In some embodiments such binding agents show binding affinities fordifferent HA polypeptides (e.g., HA polypeptides from different groups,clades, or clusters and/or from different strains) that are within 5fold binding affinity of one another. In some embodiments such bindingagents show binding affinities for different HA polypeptides that arewithin 2 fold of one another. In some embodiments such binding agentsshow binding affinities for different HA polypeptides (e.g., HApolypeptides from different groups, clades, or clusters and/or fromdifferent strains) that are within 150 fold (e.g., within 100 fold,within 50 fold, within 25 fold, within 10 fold, or within 5 fold)binding affinity of one another.

In some embodiments, such binding agents are characterized by afunctional attribute of binding to at least two of H1, H3, H5, H7,and/or H9 HA polypeptides. In some embodiments, such binding agents arecharacterized by a functional attribute of binding to at least three,four or five of the H1, H3, H5, H7, and/or H9 HA polypeptides.

In some embodiments, such binding agents are characterized by afunctional attribute of binding to at least one of subtypes H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and/or H16, and donot bind to at least one HA polypeptide of subtypes H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and/or H16. In someembodiments, such binding agents bind to HA polypeptides of subtype H1.In some embodiments, such binding agents bind to HA polypeptides ofsubtype H1 with an affinity at least 100%, at least 125%, at least 150%,at least 200% or more of that with which it binds to HA polypeptides ofat least one subtype H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15, and/or H16. In some embodiments, such binding agents bind toHA polypeptides of subtype H3. In some embodiments, such binding agentsbind to HA polypeptides of subtype H3 with an affinity at least 100%, atleast 125%, at least 150%, at least 200% or more of that with which itbinds to HA polypeptides of at least one subtype H1, H2, H4, H5, H6, H7,H8, H9, H10, H11, H12, H13, H14, H15, and/or H16.

In some embodiments, such binding agents are characterized by afunctional attribute of showing a neutralization IC₅₀ (ug/ml) within arange as described and/or exemplified herein. In some embodiments, suchbinding agents show a neutralization IC₅₀ (ug/ml) whose lower bound isabout 0.1 ug/ml and upper bound is about 10 ug/ml. In some embodiments,such binding agents show a neutralization IC₅₀ (ug/ml) whose lower boundis selected from the group consisting of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 or more ug/ml, and whose upper boundis higher than the lower bound and is selected from the group consistingof 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 or more ug/ml.

In some embodiments, such provided binding agent shows binding toinfluenza HA (e.g., group 1 and/or group 2 subtypes) with a K_(D) (nM)less than 2000 nM, less than 1500 nM, less than 1000 nM, less than 500nM, less than 250 nM, less than 225 nM, less than 200 nM, less than 175nM, less than 150 nM, less than 125 nM, less than 100 nM, less than 75nM, or less than 50 nM.

In some embodiments, such provided binding agent shows binding toinfluenza HA with a K_(a) (M⁻¹s⁻¹) whose lower bound is about 0.01×10⁵M⁻¹s⁻¹ and upper bound is about 1.0×10⁶ M⁻¹s⁻¹. such provided antibodiesshow binding to influenza HA with a K_(a) (M⁻¹s⁻¹) whose lower bound isselected from the group consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵,0.04×10⁵, 0.08×10⁵, 0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵,1.0×10⁵, 1.2×10⁵, 1.4×10⁵, 1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more M⁻¹s⁻¹,and whose upper bound is higher than the lower bound and is selectedfrom the group consisting of 1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵,3.0×10⁵, 3.5×10⁵, 4.5×10⁵, 5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵,7.5×10⁵, 8.0×10⁵, 8.5×10⁵, 9.0×10⁵, 9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶,1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or moreM⁻¹s⁻¹.

In some embodiments, such provided binding agent shows binding toinfluenza HA with a K_(d) (s⁻¹) whose lower bound is about 0.01×10⁵ s⁻¹and upper bound is about 1.0×10⁶ s⁻¹. such provided antibodies showbinding to influenza HA with a K_(a) (s⁻¹) whose lower bound is selectedfrom the group consisting of 0.01×10⁵, 0.02×10⁵, 0.04×10⁵, 0.04×10⁵,0.08×10⁵, 0.1×10⁵, 0.2×10⁵, 0.4×10⁵, 0.6×10⁵, 0.8×10⁵, 1.0×10⁵, 1.2×10⁵,1.4×10⁵, 1.6×10⁵, 1.8×10⁵, 2.0×10⁵, or more s⁻¹, and whose upper boundis higher than the lower bound and is selected from the group consistingof 1.0×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵, 3.0×10⁵, 3.5×10⁵, 4.5×10⁵,5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵, 7.5×10⁵, 8.0×10⁵, 8.5×10⁵,9.0×10⁵, 9.5×10⁵, 1.0×10⁶ 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶,1.6×10⁶, 1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or more s⁻¹.

In some embodiments, such structural features of such certain bindingagents include one or more CDRs or one or more FRs at least 65, 70, 75,80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%identical in sequence to a corresponding CDR or FR from Tables 2 and 3(SEQ ID NO: 1-60). In some embodiments, such structural features of suchbinding agents include one or more CDRs or one or more FRs comprising atleast 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99% homology to a corresponding CDR or FR from Tables 2 and 3(SEQ ID NO: 1-60). In some embodiments, such structural features of suchbinding agents include one or more CDRs and/or one or more FRs that isidentical in sequence to a corresponding CDR or FR from Tables 2 and 3(SEQ ID NO: 1-60). In some embodiments, such structural features of suchbinding agents include CDRs and FRs that are identical in sequence tothose set forth in Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features include CDR and FRsequence elements, each of which is identical to a reference CDR or FRsequence element set forth in Table 2 and/or Table 3 (SEQ ID NOs:1-60)except that it includes one or more amino acid substitutions withrespect to that reference sequence element, where the included CDR andFR sequence elements together contain no more than 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in CDR and FRsequences, as compared with the corresponding CDR and FR referencesequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). In someembodiments, such structural features include CDR and FR sequenceelements together contain no more than 18 substitutions as compared withthe corresponding CDR and FR reference sequence elements from Tables 2and 3 (SEQ ID NO: 1-60). In some embodiments, such structural featuresinclude CDR and FR sequence elements together contain no more than 15substitutions as compared with the corresponding CDR and FR referencesequence elements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features includes a FR sequenceelement, which is identical to a reference FR sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included FR sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in FR sequence, as compared with the corresponding FRreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features includes a CDR sequenceelement, which is identical to a reference CDR sequence element setforth in Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that itincludes one or more amino acid substitutions with respect to thatreference sequence element, where the included CDR sequence elementcontains no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1 substitutions in CDR sequence, as compared with thecorresponding CDR reference sequence elements from Tables 2 and 3 (SEQID NO: 1-60).

In some embodiments, such structural features include a VH sequenceelement, which is identical to a reference VH sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included VH sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in CDR sequence, as compared with the corresponding VHreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). Insome embodiments, such structural features include a structural elementcorresponding to any one of VH-1, VH-2, VH-3, VH-4, VH-5, VH-6, VH-7,VH-8, VH-9, VH-10, VH-11, VH-12, VH-13, VH-14, VH-15, VH-16 or fragmentthereof, as compared with the corresponding VH reference sequenceelements from Tables 2 and 3 (SEQ ID NO: 1-60).

In some embodiments, such structural features include a VL sequenceelement, which is identical to a reference VL sequence element set forthin Table 2 and/or Table 3 (SEQ ID NOs:1-60) except that it includes oneor more amino acid substitutions with respect to that reference sequenceelement, where the included VL sequence element contains no more than18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1substitutions in CDR sequence, as compared with the corresponding VHreference sequence elements from Tables 2 and 3 (SEQ ID NO: 1-60). Insome embodiments, such structural features include a structural elementcorresponding to any one of VL-1, VL-2, VL-3, VL-4, VL-5, VL-6, VL-7,VL-8, VL-9, VL-10, VL-11, or fragment thereof, as compared with thecorresponding VL reference sequence elements from Tables 2 and 3 (SEQ IDNO: 1-60).

In some embodiments, such structural features include a structuralelement corresponding to any one of VH-1, VH-2, VH-3, VH-4, VH-5, VH-6,VH-7, VH-8, VH-9, VH-10, VH-11, VH-12, VH-13, VH-14, VH-15, VH-16 orfragment thereof combined with any one of VL-1, VL-2, VL-3, VL-4, VL-5,VL-6, VL-7, VL-8, VL-9, VL-10, VL-11, or fragment thereof. In someembodiments, VH-1 (SEQ ID NO:1) is combined with VL-1 (SEQ ID NO:33).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR1sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 1 sequence element that has twoor more amino acid substitutions as compared to a reference CDR1sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 1 sequence element that has oneor more amino acid substitutions as compared to reference CDR1 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has at least two aminoacid substitutions as compared to reference CDR1 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR1 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 1 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR1 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR2sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 2 sequence element that has twoor more amino acid substitutions as compared to a reference CDR2sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 2 sequence element that has oneor more amino acid substitutions as compared to reference CDR2 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has at least two aminoacid substitutions as compared to reference CDR2 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR2 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 2 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR2 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that shows at least 65%,more than 70%, more than 75%, more than 80%, more than 85%, more than90%, more than 95%, or more than 99% identity with a reference CDR3sequence element from Tables 2 and/or 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 3 sequence element that has twoor more amino acid substitutions as compared to a reference CDR3sequence element from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ IDNOs:44-54). In some embodiments, such structural features include acomplementarity determining region (CDR) 3 sequence element that has oneor more amino acid substitutions as compared to reference CDR3 sequenceelement from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).In some embodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has at least two aminoacid substitutions as compared to reference CDR3 sequence element fromTables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has fewer than twoamino acid substitutions as compared to reference CDR3 sequence elementfrom Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54). In someembodiments, such structural features include a complementaritydetermining region (CDR) 3 sequence element that has an amino acidsequence that is identical to that of one of the reference CDR3 sequenceelements from Tables 2 and 3 (SEQ ID NOs:17-18 and/or SEQ ID NOs:44-54).

In some embodiments, such structural features include a VH frameworkregion sequence element that shows more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 99% percent identity with a reference VH framework regionsequence element from Table 2 (SEQ ID NOs:1-16).

In some embodiments, such structural features include a VL frameworkregion sequence element that shows more than 65%, more than 70%, morethan 75%, more than 80%, more than 85%, more than 90%, more than 95%, ormore than 99% percent identity with a reference VL framework regionsequence element from Table 3 (SEQ ID NOs:33-43).

Exemplary Binding Agents

Antibody and/or Antibody Fragment

As used herein, a binding agent refers to an agent that is capable ofbinding to an antigen or biological target. In some embodiments, aprovided binding agent is or comprises a polypeptide. In someembodiments, a provided binding agent is or comprises an antibody orfragment thereof. In some embodiments, a provided binding agent is orcomprises a monoclonal antibody or fragment thereof. In someembodiments, a provided binding agent is or comprises a polyclonalantibody or fragment thereof. In some embodiments, the binding agent isor comprises a “full length” antibody, in that it contains two heavychains and two light chains, optionally associated by disulfide bonds asoccurs with naturally-produced antibodies. In some embodiments, thebinding agent is or comprises a fragment of a full-length antibody inthat is contains some, but not all of the sequences found in afull-length antibody. For example, in some embodiments, the bindingagent is or comprises antibody fragments which include, but are notlimited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody, and Fdfragments. In some embodiments, a provided binding agent is or comprisesan antibody that is a member of an antibody class selected from thegroup consisting of IgG, IgM, IgA, IgD, IgE or fragment thereof. In someembodiments, a provided binding agent is or comprises an antibodyproduced by chemical synthesis. In some embodiments, a provided bindingagent is or comprises an antibody produced by a cell. In someembodiments, a provided binding agent is or comprises an antibodyproduced using a recombinant cell culture system. In some embodiments, aprovided binding agent is or comprises a chimeric antibody, for examplefrom mouse, rat, horse, pig, or other species, bearing human constantand/or variable region domains.

In some embodiments, a binding agent includes one or more antibodyfragments, including, but not limited to Fab′, Fab, F(ab′)2, singledomain antibodies (DABs), Fv, scFv (single chain Fv), polypeptides withantibody CDRs, scaffolding domains that display the CDRs (e.g.,anticalins) or nanobodies. For example, a provided antibody may be a VHH(i.e., an antigen-specific VHH) antibody that comprises only a heavychain. Such antibody molecules can be derived from a llama or othercamelid antibody (e.g., a camelid IgG2 or IgG3, or a CDR-displayingframe from such camelid Ig) or from a shark antibody. In someembodiments the binding agent is or comprises an avibody (diabody,tribody, tetrabody). Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

In some embodiments, provided binding agents include one or more“Mini-antibodies” or “minibodies”. Minibodies are sFv polypeptide chainswhich include oligomerization domains at their C-termini, separated fromthe sFv by a hinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating α-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B:120-126.

Peptidomimetic

In some embodiments, provided binding agents include one or moreantibody-like binding peptidomimetics. Liu et al. Cell Mol Biol(Noisy-le-grand). 2003 March; 49(2):209-16 describe “antibody likebinding peptidomimetics” (ABiPs), which are peptides that act aspared-down antibodies and have certain advantages of longer serumhalf-life as well as less cumbersome synthesis methods. Likewise, insome aspects, antibody-like molecules are cyclic or bicyclic peptides.For example, methods for isolating antigen-binding bicyclic peptides(e.g., by phage display) and for using the such peptides are provided inU.S. Patent Publn. No. 20100317547, incorporated herein by reference.

Scaffold Protein

In some embodiments, provided binding agents include one or moreantibody-like binding scaffold proteins. For example, in someembodiments, one or more CDRs arising from an antibody may be graftedonto a protein scaffold. In general, protein scaffolds may meet thegreatest number of the following criteria: (Skerra A., J. Mol. Recogn.,2000, 13:167-187): good phylogenetic conservation; knownthree-dimensional structure (as, for example, by crystallography, NMRspectroscopy or any other technique known to a person skilled in theart); small size; few or no post-transcriptional modifications; and/oreasy to produce, express and purify. The origin of such proteinscaffolds can be, but is not limited to, fibronectin (e.g., fibronectintype III domain 10), lipocalin, anticalin (Skerra A., J. Biotechnol.,2001, 74(4):257-75), protein Z arising from domain B of protein A ofStaphylococcus aureus, thioredoxin A or proteins with a repeated motifsuch as the “ankyrin repeat” (Kohl et al., PNAS, 2003, vol. 100, No. 4,1700-1705), the “armadillo repeat”, the “leucine-rich repeat” and the“tetratricopeptide repeat”. For example, anticalins or lipocalinderivatives are described in US Patent Publication Nos. 20100285564,20060058510, 20060088908, 20050106660, and PCT Publication No.WO2006/056464, incorporated herein by reference. Scaffolds derived fromtoxins such as, for example, toxins from scorpions, insects, plants,mollusks, etc., and the protein inhibitors of neuronal NO synthase (PIN)may also be used in accordance with the present invention.

Mimotope

In some embodiments, provided binding agents include a mimotope, whichcan be used to disrupt the interaction between an influenza virus andthe HA polypeptide receptor. In some embodiment, the mimotope is used toelicit an antibody response identical or similar to the that elicited byits corresponding target epitope. In some embodiments, the targetepitope is a sequence that is conserved across more than one influenzasubtype. In some embodiment, the conserved epitope is a sequence that isconserved across influenza types 1 and 2. For example, an HA sequencesfrom all influenza subtypes located within the HA-1 (head) and HA-2(stalk) domains. In some embodiments, the epitope is a conservedsequence located within the HA-1/HA-2 interface membrane proximalepitope region (MPER). In some embodiments, the epitope is a conservedsequence located within the canonical α-helix and/or residues in itsvicinity. In some embodiments, a mimotope is a peptide. In someembodiments, a mimotope is a small molecule, carbohydrate, lipid, ornucleic acid. In some embodiments, mimotopes are peptide or non-peptidemimotopes of conserved influenza epitopes. In some embodiments, bymimicking the structure of a defined viral epitope, a mimotopeinterferes with the ability of influenza virus particles to bind to itsnatural binding partners, e.g., by binding to the natural bindingpartner itself.

Stapled Peptide

In some embodiments, the provided binding agent is a stapled peptide. Insome embodiments, the stapled peptide comprises an amino acid sequencesencoding one or more CDRs and/or FRs comprising at least greater than65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99% homology and/or identity with the corresponding CDRs and/or FRsof anti-HA antibodies from Tables 2 and 3 as discussed below. In someembodiments, the stapled peptide comprises an amino acid sequenceencoding one or more VH and/or VL chain sequence comprising at leastgreater than 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98 or 99% homology and/or identity with the corresponding VH andVL chains of anti-HA antibodies from Tables 2 and 3 as discussed below.

Nucleic Acid

In certain embodiments, a binding agent is or comprise a nucleic acid,such as DNA or RNA. In certain embodiments, nucleic acids can be DNA orRNA, and can be single stranded or double-stranded. In some embodiments,nucleic acids may include one or more non-natural nucleotides; In someembodiments, nucleic acids include only natural nucleotides. In someembodiments the nucleic acid is designed to mimic an epitope within ahemagglutinin (HA) polypeptide. In some embodiments the nucleic acid isdesigned to mimic a conserved epitope within one or more Influenza HApolypeptide subtypes. In some embodiments, a provided binding agent isor comprises one or more oligonuclotides. In some embodiments, aprovided binding agent is or comprises one or more oligonuclotidescomprising a secondary structure such as loop, hairpin, fold orcombinations thereof. In some embodiments, a provided binding agent isor comprises one or more oligonuclotides comprising a higher ordered(tertiary or quaternary) structure. In some embodiments, a providedbinding agent is or comprises an aptamer.

Targeted Binding

In some embodiments a binding agent is or comprises an agent that bindsto a selected binding site. In some embodiments, such a binding agent isan engineered or designed polypeptide. In some embodiments, such aselected binding site is within a hemagglutinin (HA) polypeptide. Insome embodiments, such a selected binding site is within the MPER regionof an HA polypeptide. In some embodiments, such a selected binding agentis capable of binding to a selected binding site within an HApolypeptide MPER region independent of its glycosylation. For example,in some embodiments, binding agents are designed to be of appropriatesize that their binding to an MPER region is not prevented by itsglycosylation. In some embodiments, a binding agent binds to aglycosylated MPER region with an affinity that is at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90% or more of its affinity for an otherwise identical non-glycosylatedMPER region. In some embodiments, the binding agents bind to an HApolypeptide sequence located within the HA-1 (head) and/or HA-2 (stalk)domains. In some embodiments, the binding agents bind to an HApolypeptide sequence located within the HA-1/HA-2 interface membraneproximal epitope region (MPER). In some embodiment, the binding agentsbind to an HA polypeptide sequence located within the canonical α-helixand/or residues in its vicinity.

In some embodiments, binding agents bind to their selected binding sitesby interaction with one or more target residues. In some embodiments,such target residues are amino acids, saccharides, lipids orcombinations thereof. In some embodiments the present invention providesbinding agents that bind to an HA polypeptide, N-linked glycans on an HApolypeptide, an HA receptor, sialylated glycans on an HA receptor orvarious combinations thereof. In some embodiments, a binding agent thatbinds to an HA receptor interacts with one or more glycans on the HAreceptor. In some embodiments, binding agents bind sialylated glycans.In some embodiments, binding agents compete with influenza virus forbinding to HA receptors. In some embodiments, binding agents competewith influenza virus for binding such that binding between the influenzavirus and the HA receptor is reduced at least 1.5 fold, at least 2 fold,at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, atleast 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, atleast 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, atleast 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, atleast 19 fold, or at least 20 fold. In some embodiments, binding agentscompete with influenza virus for binding to glycans on HA receptors.

In many embodiments, binding agents have a length that is less thanabout 1000 amino acids. In some embodiments, binding agents have alength that is less than a maximum length of about 1000, 975, 950, 925,900, 875, 850, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575,550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 240,230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 125, 120, 115,110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30,25, or 20 amino acids in length. In some embodiments, binding agentshave a length that is greater than a minimum length of about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 ormore amino acids in length. In some embodiments, binding agents have alength between any one of such minimum lengths and any one of suchmaximum lengths, so long as the maximum length is longer than theminimum length. In some particular embodiments, a binding agent has alength between about 20 and 500, or between 30 and 400, or between 40and 300, or between 80 and 250 amino acids. In some embodiments, abinding agent has a length of about 84, 88, 93, 95, 98, 104, 106, 110,111, 116, 119, 123, 124, 132, 212, 215, 244, or 245.

Binding Agent Modification

In some embodiments, binding agents are comprised of natural aminoacids. In other embodiments, binding agents comprise one or moreunnatural amino acids. In some embodiments, binding agents are comprisedof combinations of natural and unnatural amino acids. In someembodiments, a binding agent is comprised of one, two or morepolypeptide chains that are covalently or non-covalently associated. Insome embodiments, a binding agent may be linked to, or part of, a longerpolypeptide chain, so long as the binding agent retains itsthree-dimensional structure and arrangement for interaction. In someembodiments, binding agents may be appended to the N- or C-termini ofanother polypeptide sequence that is or is not a binding agent. In someembodiments, binding agents are incorporated into the sequence ofanother polypeptide that is or is not a binding agent, therebyseparating the polypeptide sequence into two or more segments.

In some embodiments, appending the binding agent to the N or C terminior within the sequence of another polypeptide that is or is not abinding may allow for at least one or more of the following: a decreasein immunogenicity, increased circulation lifetime, slower in vivodegradation, inciting local immune response, interaction with the immunesystem molecules, an increase in volume, an increase in affinity for thebinding agent target(s), an increase in specificity for the bindingtarget(s), or the use of other commonly used therapeutic/prophylacticdelivery protocols. In some embodiments, appending a binding agent tothe N or C termini or within the sequence of another polypeptide that isor is not a binding agent does not have a direct effect on binding of abinding agent to a target (e.g., an HA polypeptide, the MPER region ofan HA polypeptide, N-glycans on an HA polypeptide, HA receptors orsialylated glycans on HA receptors).

Binding Agent Conjugates

In some embodiments, a provided binding agent is or comprises aconjugate, in which a binding agent moiety (comprises or consists of thebinding agent or a functional portion thereof) with a conjugated moiety.In some particular embodiments, binding agents as described herein areprovided and/or utilized in association with one or more active agentsor “payloads”, such as a therapeutic or detection agent. In some suchembodiments, association between the binding agent and the active agentand/or payload comprises at least one covalent interaction so that abinding-agent conjugate is provided.

In some embodiments, a therapeutic payload agent is an effector entityhaving a desired activity, e.g., anti-viral activity, anti-inflammatoryactivity, cytotoxic activity, etc. Therapeutic agents can be or compriseany class of chemical entity including, for example, proteins,carbohydrates, lipids, nucleic acids, small organic molecules,non-biological polymers, metals, ions, radioisotopes, etc. In someembodiments, therapeutic agents for use in accordance with the presentinvention may have a biological activity relevant to the treatment ofone or more symptoms or causes of influenza infection (e.g., forexample, anti-viral, pain-relief, anti-inflammatory, immunomodulatory,sleep-inducing activities, etc). In some embodiments, therapeutic agentsfor use in accordance with the present invention have one or more otheractivities.

In some embodiments, a payload detection agent is or comprises anymoiety which may be detected using an assay, for example due to itsspecific functional properties and/or chemical characteristics.Non-limiting examples of such agents include enzymes, radiolabels,haptens, fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

Many appropriate payload detection agents are known in the art, as aresystems for their attachment to binding agents (see, for e.g., U.S. Pat.Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein byreference). Examples of such payload detection agents includeparamagnetic ions, radioactive isotopes, fluorochromes, NMR-detectablesubstances, X-ray imaging agents, among others. For example, in someembodiments, a paramagnetic ion is one or more of chromium (III),manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper(II), neodymium (III), samarium (III), ytterbium (III), gadolinium(III), vanadium (II), terbium (III), dysprosium (III), holmium (III),erbium (III), lanthanum (III), gold (III), lead (II), and/or bismuth(III).

In some embodiments, a radioactive isotope is one or more ofastatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt,copper67, 152Eu, gallium67, 3hydrogen, iodine123, iodine125, iodine131,indium111, 59iron, 32phosphorus, radium223, rhenium186, rhenium188,75selenium, 35sulphur, technicium99m, thorium227 and/or yttrium90.Radioactively labeled antibodies may be produced according to well-knownmethods in the art. For instance, monoclonal antibodies can be iodinatedby contact with sodium and/or potassium iodide and a chemical oxidizingagent such as sodium hypochlorite, or an enzymatic oxidizing agent, suchas lactoperoxidase. Provided binding agents may be labeled withtechnetium99m by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column. In someembodiments, provided binding agent are labeled using direct labelingtechniques, e.g., by incubating pertechnate, a reducing agent such asSNCl₂, a buffer solution such as sodium-potassium phthalate solution,and the antibody. Intermediary functional groups which are often used tobind radioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

In some embodiments, a fluorescent label is or comprises one or more ofAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed, among others.

Several methods are known in the art for the attachment or conjugationof a binding agent to a payload. Some attachment methods involve the useof a metal chelate complex employing, for example, an organic chelatingagent such a diethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S.Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Provided binding agents may also be reacted with an enzymein the presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate.

Production of Anti-Influenza Antibodies

Provided antibodies, and/or characteristic portions thereof, or nucleicacids encoding them, may be produced by any available means. Methods forgenerating antibodies (e.g., monoclonal antibodies and/or polyclonalantibodies) are well known in the art. It will be appreciated that awide range of animal species can be used for the production of antisera,including rabbit, mouse, rat, hamster, guinea pig or goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art. Itwill be appreciated that antibodies can also be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

Provided antibodies (or characteristic portions) may be produced, forexample, by utilizing a host cell system engineered to express aninventive antibody-encoding nucleic acid. Alternatively or additionally,provided antibodies may be partially or fully prepared by chemicalsynthesis (e.g., using an automated peptide synthesizer).

Exemplary sources for antibody preparations suitable for the inventioninclude, but are not limited to, conditioned culture medium derived fromculturing a recombinant cell line that expresses a protein of interest,or from a cell extract of, e.g., antibody-producing cells, bacteria,fungal cells, insect cells, transgenic plants or plant cells, transgenicanimals or animal cells, or serum of animals, ascites fluid, hybridomaor myeloma supernatants. Suitable bacterial cells include, but are notlimited to, Escherichia coli cells. Examples of suitable E. coli strainsinclude: HB101, DH5α, GM2929, JM109, KW251, NM538, NM539, and any E.coli strain that fails to cleave foreign DNA. Suitable fungal host cellsthat can be used include, but are not limited to, Saccharomycescerevisiae, Pichia pastoris and Aspergillus cells. Suitable insect cellsinclude, but are not limited to, S2 Schneider cells, D. Mel-2 cells,SF9, SF21, High-5™, Mimic™-SF9, MG1 and KC1 cells. Suitable exemplaryrecombinant cell lines include, but are not limited to, BALB/c mousemyeloma line, human retinoblasts (PER.C6), monkey kidney cells, humanembryonic kidney line (293), baby hamster kidney cells (BHK), Chinesehamster ovary cells (CHO), mouse sertoli cells, African green monkeykidney cells (VERO-76), human cervical carcinoma cells (HeLa), caninekidney cells, buffalo rat liver cells, human lung cells, human livercells, mouse mammary tumor cells, TRI cells, MRC 5 cells, FS4 cells, andhuman hepatoma line (Hep G2).

Antibodies of interest can be expressed using various vectors (e.g.,viral vectors) known in the art and cells can be cultured under variousconditions known in the art (e.g., fed-batch). Various methods ofgenetically engineering cells to produce antibodies are well known inthe art. See e.g. Ausabel et al., eds. (1990), Current Protocols inMolecular Biology (Wiley, New York).

Provided antibodies may be purified, if desired, using filtration,centrifugation and/or various chromatographic methods such as HPLC oraffinity chromatography. In some embodiments, fragments of providedantibodies are obtained by methods which include digestion with enzymes,such as pepsin or papain, and/or by cleavage of disulfide bonds bychemical reduction.

Also, it will be appreciated by those of ordinary skill in the art thatpolypeptides, and particularly antibodies as described herein, may begenerated, identified, isolated, and/or produced by culturing cells ororganisms that produce antibodies (whether alone or as part of acomplex, including as part of a virus particle or virus), underconditions that allow ready screening and/or selection of polypeptidescapable of binding to influenza antigens (e.g., influenza HA). To givebut one example, in some embodiments, it may be useful to produce and/orstudy a collection of antibodies under conditions that reveal and/orfavor those variants that bind to HA polypeptides (e.g., with particularspecificity and/or affinity). In some embodiments, such a collection ofantibodies results from evolution in nature. In some embodiments, such acollection of antibodies results from engineering. In some embodiments,such a collection of antibodies results from a combination ofengineering and natural evolution.

It will be appreciated that provided antibodies may be engineered,produced, and/or purified in such a way as to improve characteristicsand/or activity of the antibody. For example, improved characteristicsof provided antibodies include, but are not limited to, increasedstability, improved binding affinity and/or avidity, increased bindingspecificity, increased production, decreased aggregation, decreasednonspecific binding, among others.

Nucleic Acids

In certain embodiments, the present invention provides nucleic acidswhich encode an antibody or a characteristic or biologically activeportion of an antibody. In some embodiments, the invention providesnucleic acids which are complementary to nucleic acids which encode anantibody or a characteristic or biologically active portion of anantibody.

In some embodiments, the invention provides nucleic acid molecules whichhybridize to nucleic acids encoding an antibody or a characteristic orbiologically active portion of an antibody. Such nucleic acids can beused, for example, as primers or as probes. To give but a few examples,such nucleic acids can be used as primers in polymerase chain reaction(PCR), as probes for hybridization (including in situ hybridization),and/or as primers for reverse transcription-PCR (RT-PCR).

In certain embodiments, nucleic acids can be DNA or RNA, and can besingle stranded or double-stranded. In some embodiments, nucleic acidsmay include one or more non-natural nucleotides; In some embodiments,nucleic acids include only natural nucleotides.

Systems for Identifying and/or Characterizing Binding Agents

The present invention provides a variety of systems for testing,characterizing, and/or identifying influenza binding agents (e.g.,anti-HA antibodies). In some embodiments, provided binding agents areused to identify and/or to characterize other influenza agents (e.g.,antibodies, polypeptides, small molecules, etc.).

In some embodiments, provided binding agents are characterized by suchsystems and methods that involve contacting the binding agent with oneor more candidate substrates, such as regions of HA polypeptides,N-glycans on HA polypeptides, HA receptors, sialylated HA receptors,glycans on sialylated HA receptors and/or umbrella topology glycans onsialylated HA receptors.

In some embodiments, an agent and/or candidate substrate may be free insolution, fixed to a support, and/or expressed in and/or on the surfaceof a cell. The candidate substrate and/or agents may be labeled, therebypermitting detection of binding. Either the agent or the candidatesubstrate is the labeled species. Competitive binding formats may beperformed in which one of the substances is labeled, and one may measurethe amount of free label versus bound label to determine the effect onbinding.

In some embodiments, binding assays involve, for example, exposing acandidate substrate to an agent and detecting binding between thecandidate substrate and the agent. A binding assay may be conducted invitro (e.g., in a candidate tube, comprising substantially only thecomponents mentioned; in cell-free extracts; and/or in substantiallypurified components). Alternatively or additionally, binding assays maybe conducted in cyto and/or in vivo (e.g., within a cell, tissue, organ,and/or organism; described in further detail below).

In certain embodiments, at least one agent is contacted with at leastone candidate substrate and an effect detected. In some embodiments, forexample, an agent is contacted with a candidate substrate, and bindingbetween the two entities is monitored. In some embodiments, an assay mayinvolve contacting a candidate substrate with a characteristic portionof an agent. Binding of the agent to the candidate substrate isdetected. It will be appreciated that fragments, portions, homologs,variants, and/or derivatives of agents may be employed, provided thatthey comprise the ability to bind one or more candidate substrates.

Binding of an agent to the candidate substrate may be determined by avariety of methods well-known in the art. The present invention providesassays involving solid phase-bound agents and detecting theirinteractions with one or more candidate substrates. Thus, an agent maycomprise a detectable marker, such as a radioactive, fluorescent, and/orluminescent label. Furthermore, candidate substrate can be coupled tosubstances which permit indirect detection (e.g. by means of employingan enzyme which uses a chromogenic substrate and/or by means of bindinga detectable antibody). Changes in the conformation of agents as theresult of an interaction with a candidate substrate may be detected, forexample, by the change in the emission of the detectable marker.Alternatively or additionally, solid phase-bound protein complexes maybe analyzed by means of mass spectrometry.

In some embodiments, the agent can be non-immobilized. In someembodiments, the non-immobilized component may be labeled (with forexample, a radioactive label, an epitope tag, an enzyme-antibodyconjugate, etc.). Alternatively or additionally, binding may bedetermined by immunological detection techniques. For example, thereaction mixture may be subjected to Western blotting and the blotprobed with an antibody that detects the non-immobilized component.Alternatively or additionally, enzyme linked immunosorbent assay (ELISA)may be utilized to assay for binding.

In certain embodiments, cells may be directly assayed for bindingbetween agents and candidate substrates. Immunohistochemical techniques,confocal techniques, and/or other techniques to assess binding are wellknown to those of skill in the art. Various cell lines may be utilizedfor such screening assays, including cells specifically engineered forthis purpose. Examples of cells used in the screening assays includemammalian cells, fungal cells, bacterial cells, or viral cells. A cellmay be a stimulated cell, such as a cell stimulated with a growthfactor. One of skill in the art would understand that the inventiondisclosed herein contemplates a wide variety of in cyto assays formeasuring the ability of agents to bind to candidate substrates.

Depending on the assay, cell and/or tissue culture may be required. Acell may be examined using any of a number of different physiologicassays. Alternatively or additionally, molecular analysis may beperformed, including, but not limited to, western blotting to monitorprotein expression and/or test for protein-protein interactions; massspectrometry to monitor other chemical modifications; etc.

In some embodiments, a binding assays described herein may be performedusing a range of concentrations of agents and/or candidate substrates.In some embodiments, the binding assays described herein are used toassess the ability of a candidate substrate to bind to an agent overrange of antibody concentrations (e.g. greater than about 100 μg/ml,about 100 μg/ml, about 50 μg/ml, about 40 μg/ml, about 30 μg/ml, about20 μg/ml, about 10 μg/ml, about 5 μg/ml, about 4 μg/ml, about 3 μg/ml,about 2 μg/ml, about 1.75 μg/ml, about 1.5 μg/ml, about 1.25 μg/ml,about 1.0 μg/ml, about 0.9 μg/ml, about 0.8 μg/ml, about 0.7 μg/ml,about 0.6 μg/ml, about 0.5 μg/ml, about 0.4 μg/ml, about 0.3 μg/ml,about 0.2 μg/ml, about 0.1 μg/ml, about 0.05 μg/ml, about 0.01 μg/ml,and/or less than about 0.01 μg/ml).

In some embodiments, any of the binding studies described herein can beexecuted in a high throughput fashion. Using high throughput assays, itis possible to screen up to several thousand agents in a single day. Insome embodiments, each well of a microtiter plate can be used to run aseparate assay against a selected candidate substrate, or, ifconcentration and/or incubation time effects are to be observed, every5-10 wells can test a single candidate substrate. Thus, a singlestandard microtiter plate can assay up to 96 binding interactionsbetween agents and candidate substrates; if 1536 well plates are used,then a single plate can assay up to 1536 binding interactions betweenagents and candidate substrates; and so forth. It is possible to assaymany plates per day. For example, up to about 6,000, about 20,000, about50,000, or more than about 100,000 assay screens can be performed onbinding interactions between antibodies and candidate substrates usinghigh throughput systems in accordance with the present invention.

In some embodiments, such methods utilize an animal host. As usedherein, an “animal host” includes any animal model suitable forinfluenza research. For example, animal hosts suitable for the inventioncan be any mammalian hosts, including primates, ferrets, cats, dogs,cows, horses, rodents such as, mice, hamsters, rabbits, and rats. Incertain embodiments, an animal host used for the invention is a ferret.In particular, in some embodiments, an animal host is naïve to viralexposure or infection prior to administration of an agent (optionally inan inventive composition). In some embodiments, the animal host isinoculated with, infected with, or otherwise exposed to virus prior toor concurrent with administration of an agent. An animal host used inthe practice of the present invention can be inoculated with, infectedwith, or otherwise exposed to virus by any method known in the art. Insome embodiments, an animal host may be inoculated with, infected with,or exposed to virus intranasally.

In some embodiments, a suitable animal host may have a similardistribution of umbrella vs. cone topology glycans and/or α2,6 glycansvs. α2,3 glycans to the distribution found in the human respiratorytract. For example, it is contemplated that a ferret as an animal hostmay be more representative than a mouse when used as model of diseasecaused by influenza viruses in humans (Tumpey, et al. Science (2007)315; 655-659). Without wishing to be bound any theories, the presentinvention encompasses the idea that ferrets may have a more similardistribution of glycans in the respiratory tract to those in the humanrespiratory tract than mouse does to human.

Naïve and/or inoculated animals may be used for any of a variety ofstudies. For example, such animal models may be used for virustransmission studies as in known in the art. It is contemplated that theuse of ferrets in virus transmission studies may serve as a reliablepredictor for virus transmission in humans. For example, airtransmission of viral influenza from inoculated animals (e.g., ferrets)to naïve animals is known in the art (Tumpey, et al. Science (2007) 315;655-659). Virus transmission studies may be used to test agents. Forexample, agents may be administered to a suitable animal host before,during or after virus transmission studies in order to determine theefficacy of said agent in blocking virus binding and/or infectivity inthe animal host. Using information gathered from virus transmissionstudies in an animal host, one may predict the efficacy of an agent inblocking virus binding and/or infectivity in a human host.

Pharmaceutical Compositions

The present invention provides compositions comprising one or moreprovided binding agents. In some embodiments the present inventionprovides at least one binding agent and at least one pharmaceuticallyacceptable excipient. Such pharmaceutical compositions may optionallycomprise and/or be administered in combination with one or moreadditional therapeutically active substances. In some embodiments,provided pharmaceutical compositions are useful in medicine. In someembodiments, provided pharmaceutical compositions are useful asprophylactic agents (i.e., vaccines) in the treatment or prevention ofinfluenza infection or of negative ramifications and/or symptomsassociated or correlated with influenza infection. In some embodiments,provided pharmaceutical compositions are useful in therapeuticapplications, for example in individuals suffering from or susceptibleto influenza infection. In some embodiments, pharmaceutical compositionsare formulated for administration to humans.

For example, pharmaceutical compositions provided here may be providedin a sterile injectable form (e.g., a form that is suitable forsubcutaneous injection or intravenous infusion). For example, in someembodiments, pharmaceutical compositions are provided in a liquid dosageform that is suitable for injection. In some embodiments, pharmaceuticalcompositions are provided as powders (e.g. lyophilized and/orsterilized), optionally under vacuum, which are reconstituted with anaqueous diluent (e.g., water, buffer, salt solution, etc.) prior toinjection. In some embodiments, pharmaceutical compositions are dilutedand/or reconstituted in water, sodium chloride solution, sodium acetatesolution, benzyl alcohol solution, phosphate buffered saline, etc. Insome embodiments, powder should be mixed gently with the aqueous diluent(e.g., not shaken).

In some embodiments, provided pharmaceutical compositions comprise oneor more pharmaceutically acceptable excipients (e.g., preservative,inert diluent, dispersing agent, surface active agent and/or emulsifier,buffering agent, etc.). In some embodiments, pharmaceutical compositionscomprise one or more preservatives. In some embodiments, pharmaceuticalcompositions comprise no preservative.

In some embodiments, pharmaceutical compositions are provided in a formthat can be refrigerated and/or frozen. In some embodiments,pharmaceutical compositions are provided in a form that cannot berefrigerated and/or frozen. In some embodiments, reconstituted solutionsand/or liquid dosage forms may be stored for a certain period of timeafter reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days,7 days, 10 days, 2 weeks, a month, two months, or longer).

Liquid dosage forms and/or reconstituted solutions may compriseparticulate matter and/or discoloration prior to administration. In someembodiments, a solution should not be used if discolored or cloudyand/or if particulate matter remains after filtration.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In some embodiments, such preparatory methods include thestep of bringing active ingredient into association with one or moreexcipients and/or one or more other accessory ingredients, and then, ifnecessary and/or desirable, shaping and/or packaging the product into adesired single- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to a dose which would be administered to asubject and/or a convenient fraction of such a dose such as, forexample, one-half or one-third of such a dose.

Relative amounts of active ingredient, pharmaceutically acceptableexcipient, and/or any additional ingredients in a pharmaceuticalcomposition in accordance with the invention may vary, depending uponthe identity, size, and/or condition of the subject treated and/ordepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

Pharmaceutical compositions of the present invention may additionallycomprise a pharmaceutically acceptable excipient, which, as used herein,may be or comprise solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21st Edition,A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md., 2006)discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention.

Vaccines

In some embodiments, the present invention provides vaccine compositionsfor use, and/or for exam in passive immunization (i.e., immunizationwherein a binding agent is administered to a subject) of a subject whois suffering from or susceptible to influenza infection. In someembodiments, passive immunization occurs when antibodies are transferredfrom mother to fetus during pregnancy. In some embodiments, passiveimmunization includes administration of antibodies directly to anindividual (e.g., by injection, orally, nasally, etc.).

In some embodiments, prophylactic applications may include administeringvaccines. In some embodiments, vaccination is tailored to the individualpatient. For example, as described below, serum may be collected from apatient and tested for presence of influenza, and in some embodimentsfor one or more particular influenza subtypes. In some embodiments,appropriate recipients of provided vaccines are individuals sufferingfrom or susceptible to infection with one or more influenza subtypesbound and/or neutralized by a provided antibody.

In some embodiments, a vaccine composition comprises at least oneadjuvant. Any adjuvant may be used in accordance with the presentinvention. A large number of adjuvants are known; a useful compendium ofmany such compounds is prepared by the National Institutes of Health andcan be found on their website. See also Allison (1998, Dev. Biol.Stand., 92:3-11; incorporated herein by reference), Unkeless et al.(1998, Annu. Rev. Immunol., 6:251-281; incorporated herein byreference), and Phillips et al. (1992, Vaccine, 10:151-158; incorporatedherein by reference). Hundreds of different adjuvants are known in theart and could be employed in the practice of the present invention.Exemplary adjuvants that can be utilized in accordance with theinvention include, but are not limited to, cytokines, gel-type adjuvants(e.g., aluminum hydroxide, aluminum phosphate, calcium phosphate, etc.);microbial adjuvants (e.g., immunomodulatory DNA sequences that includeCpG motifs; endotoxins such as monophosphoryl lipid A; exotoxins such ascholera toxin, E. coli heat labile toxin, and pertussis toxin; muramyldipeptide, etc.); oil-emulsion and emulsifier-based adjuvants (e.g.,Freund's Adjuvant, MF59 [Novartis], SAF, etc.); particulate adjuvants(e.g., liposomes, biodegradable microspheres, saponins, etc.); syntheticadjuvants (e.g., nonionic block copolymers, muramyl peptide analogues,polyphosphazene, synthetic polynucleotides, etc.); and/or combinationsthereof. Other exemplary adjuvants include some polymers (e.g.,polyphosphazenes; described in U.S. Pat. No. 5,500,161, which isincorporated herein by reference), Q57, QS21, squalene,tetrachlorodecaoxide, etc. Pharmaceutically acceptable excipients havebeen previously described in further detail in the above sectionentitled “Pharmaceutical Compositions.”

Combination Therapy

Pharmaceutical compositions of the present invention may be administeredeither alone or in combination with one or more other therapeutic agentsincluding, but not limited to, vaccines and/or antibodies. By “incombination with,” it is not intended to imply that the agents must beadministered at the same time or formulated for delivery together,although these methods of delivery are within the scope of theinvention. In general, each agent will be administered at a dose and ona time schedule determined for that agent. Additionally, the inventionencompasses the delivery of the pharmaceutical compositions incombination with agents that may improve their bioavailability, reduceor modify their metabolism, inhibit their excretion, or modify theirdistribution within the body. Although the pharmaceutical compositionsof the present invention can be used for treatment of any subject (e.g.,any animal) in need thereof, they are most preferably used in thetreatment of humans.

In some embodiments, pharmaceutical compositions of the presentinvention may be administered in combination with one or more otheragents. In some embodiments pharmaceutical compositions of the presentinvention may be administered in combination with one or more otherpharmaceutical agents (e.g., anti-influenza vaccine, anti-viral agent,pain relievers, anti-inflammatories, antibiotics, steroidal agents,antibodies, sialydase, etc). In some embodiments, pharmaceuticalcompositions of the present invention and/or agents (e.g., antibodies)may be administered in combination with an adjuvant.

In some embodiments, pharmaceutical compositions of the presentinvention are administered in combination with one or more anti-viralagents. In some embodiments, such anti-viral agents include, but are notlimited to, acyclovir, ribavirin, amantadine, remantidine, zanamivir(Relenza), oseltamivir (Tamiflu), amantadine, rimantadine and/orcombinations thereof.

In some embodiments, pharmaceutical compositions of the presentinvention are administered in combination one or more vaccines. In someembodiments, the vaccine is a anti-viral vaccine. In some embodiments,the vaccine is an anti-influenza vaccine. In some embodiments, theanti-influenza vaccine is to treat seasonal influenza (e.g., commonlyreferred to as the “flu”). In some embodiments, the anti-influenzavaccine is the flu shot and/or FluMist. In some embodiments, theanti-influenza vaccine is targeted to a specific combination of one ormore HA polypeptides (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10,H11, H12, H13, H14, H15, or H16 polypeptides). In some embodiments, theanti-influenza vaccine is specific for one or more combinations of H1N1,H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, or H10N7 viruses. Insome embodiments, the anti-influenza vaccine is specific to H1N1viruses. In some embodiments, the anti-influenza vaccine is specific toH3N2 viruses. In some embodiments, the anti-influenza vaccine isspecific to H1N1 and H3N2 viruses.

In some embodiments pharmaceutical compositions may be administered incombination with one or more other pharmaceutical agents used to treatthe symptoms associated with influenza virus infection. In someembodiments, pharmaceutical agents used to treat the symptoms associatedwith influenza infection are pain relievers, anti-inflammatories,antibiotics and/or combinations thereof. In some embodiments,pharmaceutical agents used to treat the inflammation symptoms associatedwith influenza infection is selected from the group consisting of NSAID,Steroid, Glucocorticoid, and/or combinations thereof. In someembodiments, NSAID pharmaceutical agents used to treat the influenzasymptoms associated with influenza infection is selected from the groupconsisting of acetaminophen, ibuprofen, aspirin, naproxen and/orcombinations thereof.

Methods of Administration

Pharmaceutical compositions of the present invention can be administeredby a variety of routes, including oral, intravenous, intramuscular,intra-arterial, subcutaneous, intraventricular, transdermal,interdermal, rectal, intravaginal, intraperitoneal, topical (as bypowders, ointments, creams, or drops), mucosal, nasal, buccal, enteral,sublingual; by intratracheal instillation, bronchial instillation,and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.In general the most appropriate route of administration will depend upona variety of factors including the nature of the agent (e.g., itsstability in the environment of the gastrointestinal tract), thecondition of the patient (e.g., whether the patient is able to tolerateoral administration), etc.

At present the oral or nasal spray or aerosol route (e.g., byinhalation) is most commonly used to deliver therapeutic agents directlyto the lungs and respiratory system. However, the invention encompassesthe delivery of the inventive pharmaceutical composition by anyappropriate route taking into consideration likely advances in thesciences of drug delivery.

In some embodiments, preparations for inhaled or aerosol deliverycomprise a plurality of particles. In some embodiments, suchpreparations have a mean particle size of 4, 5, 6, 7, 8, 9, 10, 11, 12,or 13 microns. In some embodiments, preparations for inhaled or aerosoldelivery are formulated as a dry powder. In some embodiments,preparations for inhaled or aerosol delivery are formulated as a wetpowder, for example through inclusion of a wetting agent. in someembodiments, the wetting agent is selected from the group consisting ofwater, saline, or other liquid of physiological pH.

In some embodiments, inventive compositions are administered as drops tothe nasal or buccal cavity. In some embodiments, a dose may comprise aplurality of drops (e.g., 1-100, 1-50, 1-20, 1-10, 1-5, etc.)

In some embodiments, inventive compositions are administered using adevice that delivers a metered dosage of composition.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. No. 4,886,499, U.S. Pat. No. 5,190,521, U.S. Pat.No. 5,328,483, U.S. Pat. No. 5,527,288, U.S. Pat. No. 4,270,537, U.S.Pat. No. 5,015,235, U.S. Pat. No. 5,141,496, U.S. Pat. No. 5,417,662.Intradermal compositions may also be administered by devices which limitthe effective penetration length of a needle into the skin, such asthose described in WO99/34850, incorporated herein by reference, andfunctional equivalents thereof. Also suitable are jet injection deviceswhich deliver liquid compositions to the dermis via a liquid jetinjector or via a needle which pierces the stratum corneum and producesa jet which reaches the dermis. Jet injection devices are described forexample in U.S. Pat. No. 5,480,381, U.S. Pat. No. 5,599,302, U.S. Pat.No. 5,334,144, U.S. Pat. No. 5,993,412, U.S. Pat. No. 5,649,912, U.S.Pat. No. 5,569,189, U.S. Pat. No. 5,704,911, U.S. Pat. No. 5,383,851,U.S. Pat. No. 5,893,397, U.S. Pat. No. 5,466,220, U.S. Pat. No.5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat.No. 5,064,413, U.S. Pat. No. 5,520,639, U.S. Pat. No. 4,596,556, U.S.Pat. No. 4,790,824, U.S. Pat. No. 4,941,880, U.S. Pat. No. 4,940,460, WO97/37705 and WO 97/13537. Also suitable are ballistic powder/particledelivery devices which use compressed gas to accelerate compositions inpowder form through the outer layers of the skin to the dermis.Additionally, conventional syringes may be used in the classical mantouxmethod of intradermal administration.

Diagnostics Applications

In some embodiments, influenza binding agents in accordance with theinvention are used for diagnostic applications. For example, by virtueof the variety of binding profiles of binding agents, diagnostic assaysmay be employed which will detect a plurality of influenza genotypesand/or subtypes, so as to provide a pan-influenza binding agent (i.e., apan-influenza antibody), while at the same time being able to dissectindividual genotypes and/or subtypes by subtractive analysis.

For diagnostic purposes, binding agents may be used in a wide variety offormats for detecting HA protein, discerning influenza genotypes and/orsubtypes, detecting virions and antibodies. For diagnostic purposes, awide variety of labels may be employed, which for the most part havebeen mentioned previously. These include, but are not limited to,fluorophores, chemiluminescent moieties, radioisotopes, enzymes,particles (e.g., colloidal carbon particles, gold particles, latexparticles, etc.) ligands for which there are high affinity receptors,and prolabels, which can be activated to provide a detectable signal.

In some embodiments, a surface is coated with a protein, which can bindto influenza antigens as free protein (e.g., circulating proteins) or aspart of an intact or partially intact virion. One may use providedbinding agents of the invention which bind to multiple influenzagenotypes and/or subtypes. In some embodiments, binding agents inaccordance with the invention bind to at least one, two, three, four,five, or more than five different genotypes and/or subtypes.

In some embodiments, assays may involve contacting a surface with amedium, which may contain free or influenza-associated protein(s), wherethe medium may be the sample or a solution of known HA of one or moregenotypes and/or subtypes. After incubation and washing to removenon-specifically bound protein, the assay may proceed in various mannersdepending upon what is being assayed. Where a blood sample suspected ofbeing seropositive is being assayed, the sample may be applied to thelayer of HA protein, incubated, and washed, and the presence of humanantibodies bound to the protein layer determined. One may use labeledα-human antibodies (other than against the isotype of the subjectantibodies, where the subject antibodies have been initially used). Inassays for antibodies in seropositive subjects, subject antibodies maybe used as controls with the same reagent used to detect any humananti-influenza antibodies in the sera of such subjects. The specificityof the antibodies in the sample can be confirmed by using the subjectantibodies, which are differentially labeled from the anti-humanantibodies and determine whether they are blocked by the antibodies inthe sample.

Where the sample is assayed for influenza HA protein, detection employslabeled subject antibodies, the selection depending upon whether one isinterested in genotyping or detection of HA protein. After washing awaynon-specifically bound antibody, the presence of labeled antibodies isdetermined by detecting the presence of the label in accordance withknown techniques. Alternatively or additionally, where the subjectantibodies are bound to a surface, a labeled lectin for HA may beemployed to detect the presence of HA protein.

Binding agents in accordance with the invention can be used to measurethe reactivity of other binding agents, including antibodies in sera,monoclonal antibodies, antibodies expressed as a result of geneticengineering, etc. In some embodiments, intact virions are used. In someembodiments, conformationally conserved envelope proteins are used.

Labeled subject antibodies may be used in assaying for the presence ofinfluenza from biopsy material. Labeled antibody may be incubated withimmobilized biopsy material, such as a lung slice, with a solution ofone or more of the subject labeled antibodies. After washing awaynon-specifically bound antibodies, the presence of the antibodies boundto the cells of the biopsied tissue may be detected in accordance withthe nature of the label.

In some embodiments, influenza binding agents in accordance with theinvention can be used to identify influenza receptors. Those skilled inthe art will appreciate the multitude of ways this can be accomplished(Sambrook J., Fritsch E. and Maniatis T. Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989; andAusubel et al., eds., Current Protocols in Molecular Biology, 1987; bothof which are incorporated herein by reference). Typically, protein andpeptide receptors can be identified by determining whether a bindingagent able to bind HA can inhibit attachment of influenza virions to acell susceptible to influenza infection. Thus, receptors for influenzaHA proteins and peptides can be identified in this manner. A susceptiblecell can be incubated in the presence of influenza and anti-influenza HAbinding agent, and a cell-binding assay can be utilized to determinewhether attachment is decreased in the presence of the binding agent.

Cells expressing putative receptors for influenza and/or libraries ofputative receptors for influenza may be screened for their abilities tobind influenza. For example, cells expressing a putative influenzareceptor (e.g., a receptor for influenza HA) can be contacted with aninfluenza protein or peptide in the presence of an antibody for a timeand under conditions sufficient to allow binding of the influenzaprotein or peptide to putative receptor on the surface of the cell.Alternatively or additionally, influenza proteins, peptides, or virionscan be pre-incubated with antibody prior to contacting the putativereceptor on the cell surface. Binding can be detected by any means knownin the art, e.g., flow cytometry etc. (see Ausubel et al. or Sambrook etal., supra). A decrease in binding to the surface of the cell in thepresence of antibody compared to binding in the absence of the cell inthe absence of the antibody indicates the identification of an influenzareceptor.

In some embodiments, methods of identifying influenza receptors (e.g.,such as HA receptors) include the use of solid supports, such as beads,columns, and the like. For example, receptors for influenza proteins andpeptides (e.g., HA proteins and/or fragments thereof) and/or influenzavirions can be identified by attaching an influenza antibody to a solidsupport and then contacting the antibody with an influenza protein orpeptide for a time sufficient for the influenza protein or peptide tobind to the antibody. This provides an influenza protein ligand forputative influenza receptors that can be contacted with theantibody:ligand complex on the solid support for a time and underconditions sufficient to allow binding of a receptor to the influenzaprotein or peptide. Proteins can be expressed from a library or providedas a cell extract or purified protein preparation from natural orrecombinant cells. Once specific binding complexes between the influenzaprotein peptide are formed, unbound influenza proteins or peptides,e.g., library proteins or peptide that did not bind specifically to theinfluenza proteins or peptides, are removed, e.g., by standard washingsteps. Bound proteins are then eluted and identified, e.g., by gelelectrophoresis.

Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods in accordance with the presentinvention. Kits typically comprise one or more influenza binding agentsin accordance with the invention. In some embodiments, kits comprise acollection of different influenza binding agents to be used fordifferent purposes (e.g., diagnostics, treatment, and/or prophylaxis).Typically kits will comprise sufficient amounts of influenza bindingagents to allow a user to perform multiple administrations to asubject(s) and/or to perform multiple experiments. In some embodiments,kits are supplied with or include one or more influenza antibodies thathave been specified by the purchaser.

In certain embodiments, kits for use in accordance with the presentinvention may include one or more reference samples; instructions (e.g.,for processing samples, for performing tests, for interpreting results,for solubilizing influenza binding agents); buffers; and/or otherreagents necessary for performing tests. In certain embodiments kits cancomprise panels of antibodies. Other components of kits may includecells, cell culture media, tissue, and/or tissue culture media.

Kits may comprise instructions for use. For example, instructions mayinform the user of the proper procedure by which to prepare apharmaceutical composition comprising influenza binding agents and/orthe proper procedure for administering pharmaceutical compositions to asubject.

In some embodiments, kits include a number of unit dosages of apharmaceutical composition comprising influenza binding agents. A memoryaid may be provided, for example in the form of numbers, letters, and/orother markings and/or with a calendar insert, designating the days/timesin the treatment schedule in which dosages can be administered. Placebodosages, and/or calcium dietary supplements, either in a form similar toor distinct from the dosages of the pharmaceutical compositions, may beincluded to provide a kit in which a dosage is taken every day.

Kits may comprise one or more vessels or containers so that certain ofthe individual components or reagents may be separately housed. Kits maycomprise a means for enclosing the individual containers in relativelyclose confinement for commercial sale, e.g., a plastic box, in whichinstructions, packaging materials such as Styrofoam, etc., may beenclosed.

In some embodiments, kits are used in the treatment, diagnosis, and/orprophylaxis of a subject suffering from and/or susceptible to influenza.In some embodiments, inventive kits comprise at least one component of adelivery device, e.g., a syringe, needle, applicator, inhaler, etc. Insome such embodiments, the invention provides a kit comprising at leastone component of a delivery device, e.g., an inhaler and/or syringe anda dose of an of an agent. In some embodiments, kits comprise (i) atleast one influenza binding agent; (ii) a syringe, needle, applicator,inhaler, etc. for administration of the at least one influenza bindingagent to a subject; and (iii) instructions for use.

In some embodiments, kits are used in the treatment, diagnosis, and/orprophylaxis of a subject suffering from and/or susceptible to influenza.In some embodiments, such kits comprise (i) at least one influenzabinding agent (i.e., a pan-influenza antibody) provided as a lyophilizedpowder; and (ii) a diluent for reconstituting the lyophilized powder.Such kits may optionally comprise a syringe, needle, applicator, etc.for administration of the at least one influenza binding agent to asubject; and/or instructions for use.

The present invention provides kits containing reagents for thegeneration of vaccines comprising at least one influenza binding agent.In some embodiments, such kits may include (i) cells expressinginfluenza binding agents, characteristic portions thereof, and/orbiologically active portions thereof; (ii) media for growing the cells;and (iii) columns, resin, buffers, tubes, and other tools useful forantibody purification. In some embodiments, such kits may include (i)plasmids containing nucleotides encoding influenza binding agents,characteristic portions thereof, and/or biologically active portionsthereof; (ii) cells capable of being transformed with the plasmids, suchas mammalian cell lines, including but not limited to, Vero and MDCKcell lines; (iii) media for growing the cells; (iv) expression plasmidscontaining no nucleotides encoding influenza binding agents as negativecontrols; (v) columns, resin, buffers, tubes, and other tools useful forantibody purification; and (vi) instructions for use.

In some embodiments, kits are used to detect the presence of influenzain one or more samples. Such samples may be pathological samples,including, but not limited to, blood, serum/plasma, peripheral bloodmononuclear cells/peripheral blood lymphocytes (PBMC/PBL), sputum,urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids,cervical smears, pus samples, food matrices, and tissues from variousparts of the body such as brain, spleen, and liver. Such samples may beenvironmental samples, including, but not limited to, soil, water, andflora. Other samples that have not been listed may also be applicable.In some embodiments, such kits comprise (i) at least one influenzabinding agent; (ii) a sample known to contain influenza, as a positivecontrol; and (iii) a sample known not to contain influenza, as anegative control; and (iv) instructions for use.

In some embodiments, kits are used to neutralize influenza in one ormore samples. Such kits may provide materials needed to treat aninfluenza-containing sample with at least one influenza binding agentand to test the ability of the treated sample to infect cultured cellsrelative to untreated sample. Such kits may include (i) at least oneinfluenza binding agent; (ii) cells capable of being cultured andinfected with influenza; (iii) binding agent that is incapable ofbinding to and neutralizing influenza, as a negative control; (iv) abinding agent that is capable of binding to and neutralizing influenza,as a positive control; (v) a sample known not to contain influenza, as anegative control; (vi) a sample known to contain influenza, as apositive control; and (vii) instructions for use.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXEMPLIFICATION Example 1: Identification and Characterization ofInfluenza Antibodies

The present Example describes production and/or testing of variousantibodies provided in accordance with the present invention.

Anti-HA monoclonal antibodies were generated and framework (FR)sequences were determined. Table 2 depicts exemplary amino acidsequences of VH domains of anti-HA antibodies. Table 3 depicts exemplaryamino acid sequences of VL domains of anti-HA antibodies.Complementarity Determining Regions (CDRs) of each of the heavy andlight chains are depicted in bold and listed in CDR1, CDR2, and CDR3columns in the Tables 2 and 3.

An exemplary antibody was characterized for binding to HA from differentsubtypes of influenza. Sequences of the exemplary antibody framework andcomplement determining regions are indicated in Table 4 below. Theexemplary antibody binds to both group 1 and group 2 subtypes of HA withdifferential binding affinity.

TABLE 4 Amino Acid Sequence of VH and VL Chains of Exemplary AntibodyExemplary Amino Acid Sequence (CDR Framework Sequences in bold) CDR 1CDR 2 CDR 3 VH EVQLLESGGGLVKPGQSLKLSCAAS GFTFTSY SYDGSYDSELRSLLYFEWLSQGYFNP GFTFTSYGMHWVRQPPGKGLEWVAV (SEQ ID NO: 17)(SEQ ID NO: 19) (SEQ ID NO: 21) ISYDGSYKYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDS ELRSLLYFEWLSQGYFNPWGAGTTL TVSSASTK(SEQ ID NO: 1) VL EIVMTQSPDSLAVSLGERATINCKS KSSQSVTYNYKNYLA WASTRESQQYYRTPPT SQSVTYNYKNYLAWYQQKPGQPPKL (SEQ ID NO: 44) (SEQ ID NO: 55)(SEQ ID NO: 59) LIYWASTRESGVPDRFSGSGSGTDF TLTISSLQAEDVAVYYCQQYYRTPPTFGGGTKLDIKGS (SEQ ID NO: 33)

The exemplary antibody was tested for binding to HA polypeptides in anin vitro binding assay. Maxisorp 96-well plate wells were coated with0.2 μg an HA polypeptide of different subtypes (H1, H3, H5, H7 and H9)and left overnight at 4° C. The HA polypeptide coated plates were washedthrice with PBS and blocked with 1% BSA in PBST. Differentconcentrations of the exemplary antibody along with C179 antibody(control) were added to HA polypeptide coated wells and the plate wasincubated at RT for 2 hrs. The plate was washed thrice with PBST and thewells containing agents were incubated with mouse-anti-6×His antibody(1:1000 dilution) for 1 hr at RT. The plates were washed thrice withPBST and all wells were incubated with goat-anti-mouse HRP antibody for1 hr at RT. Post-incubation the wells were washes with PBST and thebound HRP was measured using TMB substrate. TMB substrate was added tothe wells, incubated for 3 minutes, followed by addition of 1 N sulfuricacid. Absorbance was measured at 450 nm.

As can be seen in FIG. 2A (bottom panel) and FIG. 2B, our results showthat the exemplary antibody binds to various HA polypeptides (H1, H3,H5, H7 and H9).

Example 2. Binding Affinity Between an Exemplary Influenza Antibody andthe Targets of the Influenza Antibody

The present example shows a calculation of binding affinity, asrepresented as an equilibrium dissociation constant (K_(D)), between anexample influenza antibody and the target of the antibody. In thisexample, the antibody is an antibody of Example 1 and the targets of theantibody are HA polypeptide from different influenza strains.

Binding affinity between the exemplary antibody and an HA polypeptide isa function of the concentrations of both the antibody and the HApolypeptide. In the present example, the binding affinity isquantitatively described using equilibrium dissociation rate constant(K_(D)). An example of how to measure the dissociation constant isdescribed below.

HA polypeptide coated plates were used to perform ELISA assays with anexemplary antibody as described previously. The measured absorbance at450 nm was used to calculate the fractional saturation of the receptor.Fractional saturation was plotted as a function of molar concentrationof the antibody. Data was fit to the following equation:

$y = \frac{I_{0}}{\left( {K_{d} + I_{0}} \right)}$where y is the fractional saturation, I₀ is the concentration of theantibody and K_(D) is the equilibrium dissociation rate constant.

Using the above referenced calculation, and applying regressionanalysis, we have observed differential K_(D) values for HA polypeptidesfrom different Influenza subtypes (FIG. 2A, top panel). In someembodiments, we have observed exemplary antibodies with K_(D) values inthe range of 0.01 to 100 nM for binding of antibodies to different HApolypeptide subtypes. In some embodiments, we have observed exemplaryantibodies with K_(D) values in the range of 0.1 to 500 nM for bindingof antibodies to different HA polypeptide subtypes. In some embodiments,we have observed K_(D) values in the range of 10 to 100 nM for bindingof antibodies to HA polypeptides subtypes. In some embodiments, we haveobserved K_(D) values in the range of 50 to 100 nM for binding ofantibodies to HA polypeptides subtypes.

Example 3. Kinetic Rate Evaluation of an Influenza Antibody

The present example illustrates the ability of an exemplary influenzaantibody to reduce virus infectivity in an in vitro binding assays. Thepresent example shows an alternative method for calculating bindingaffinity, as represented as an association rate constant (k_(a)),dissociation rate constant (k_(d)), and equilibrium dissociationconstant K_(D) between an example influenza antibody and the target ofthe antibody. In this example, the antibody is an antibody of Example 1and the targets of the antibody are HA polypeptide from differentinfluenza strains.

Binding affinity between the exemplary antibody and an HA polypeptide isa function of the concentrations of both the antibody and the HApolypeptide. In the present example, binding affinity is quantitativelydescribed using association constant (k_(a)), dissociation constant(k_(d)) and equilibrium constant (K_(D)). An example of how to measureand calculate these constants is described below.

In the experiment, a Biacore™ systems was used to monitor the kineticrate interaction between the exemplary antibody and various HApolypeptides in real time. The Biacore system works on the principle ofSurface Plasmon Resonance (SPR), which is able to accurately measurechanges in refractive index at a surface. Briefly, one interactant (theligand) is immobilized to the surface of a sensor chip. A solutioncontaining potential binding partner(s) is passed over the immobilizedsurface, and binding is visualized as a change in refractive index atthe surface (response units (RU)) over time. Surface Plasmon Resonanceallows for immediate visualization of interactions in a label-freemanner, lessening the potential impact of labels on the interaction ofinterest.

The following biotinylated polypeptides; H1 (A/Solomon Islands/03/06),H3 (A/Wyoming/3/2003), H5 (A/Vietnam/1203/2004), H7(A/Netherlands/219/03) and H9 (A/Hong Kong/1073/99) were each bound tothe surface of a separate Biacore sensor chip. An antibody of varyingconcentration, was passed over the chip for SPR binding analysis. Datawas plotted as a function of response difference measured in RU versestime. The various kinetic rate values were calculated using thefollowing equations:

$\frac{d\lbrack{AB}\rbrack}{dt} = {k_{a} \cdot \lbrack A\rbrack \cdot \lbrack B\rbrack}$$\frac{- {d\lbrack{AB}\rbrack}}{dt} = {k_{d} \cdot \lbrack{AB}\rbrack}$$K_{D} = \frac{k_{d}}{k_{a}}$where d[AB] is RU, where [A] is antibody concentration, [B] is[R_(max)−R], k_(a) is the association constant, k_(d) is thedissociation constant and K_(D) is the equilibrium dissociationconstant.

Using the above referenced calculation, we have observed differentialkinetic binding rates between the antibody and HA polypeptides fromdifferent influenza subtypes (FIGS. 3A-E). In some embodiments, we haveobserved exemplary antibodies with K_(D) values in the range of 0.01 to100 nM for binding of antibodies to different HA polypeptide subtypes.In some embodiments, we have observed exemplary antibodies with K_(D)values in the range of 0.1 to 500 nM for binding of antibodies todifferent HA polypeptide subtypes. In some embodiments, we have observedK_(D) values in the range of 10 to 100 nM for binding of antibodies toHA polypeptides subtypes. In some embodiments, we have observed K_(D)values in the range of 50 to 100 nM for binding of antibodies to HApolypeptides subtypes.

In some embodiments, we have observed exemplary antibodies with k_(a)values in the range of 0.01×10⁵ to 0.1×10⁵ M⁻¹s⁻¹ for binding ofantibodies to different HA polypeptide subtypes. In some embodiments, wehave observed exemplary antibodies with k_(a) values in the range of0.1×10⁵ to 1.0×10⁵ M⁻¹s⁻¹ for binding of antibodies to different HApolypeptide subtypes. In some embodiments, we have observed exemplaryantibodies with k_(a) values in the range of 1.0×10⁵ to 1.0×10⁶ M⁻¹s⁻¹for binding of antibodies to different HA polypeptide subtypes.

In some embodiments, we have observed exemplary antibodies with k_(d)values in the range of 0.01×10⁻⁵ to 0.1×10⁻⁵s⁻¹ for binding ofantibodies to different HA polypeptide subtypes. In some embodiments, wehave observed exemplary antibodies with k_(d) values in the range of0.1×10⁻⁵ to 1.0×10⁻⁵s⁻¹ for binding of antibodies to different HApolypeptide subtypes. In some embodiments, we have observed exemplaryantibodies with k_(d) values in the range of 1.0×10⁻⁵ to 10.0×10⁻⁶s⁻¹for binding of antibodies to different HA polypeptide subtypes.

Example 4. Antibodies Inhibit Virus Infectivity In Vitro

The present example illustrates the ability of an exemplary influenzaantibody to prevent virus infectivity in an in vitro binding assay.

The ability of the exemplary influenza antibody of Example 1 to inhibitinfluenza infection was evaluated in vitro using MDCK (Madin-DarbyCanine Kidney) cells, an epithelial cell line commonly used for thepropagation and testing of influenza virus strains. The inhibitoryeffects of the antibody on infectivity were determined by measuring bothviral yield and the extent of influenza-induced cytopathic effects (CPE)on the host cells. Plaque assay and qRT-PCR were employed to quantifyviral production. A cell viability assay was used to measure CPE levels.The experiments were set up to allow for the antibody to first bind toits viral target during a one hour pre-incubation period beforeintroduction to the host cells. Infection was carried out in thepresence of low levels of trypsin (1 μM). The plaque assay was performedby inoculating confluent monolayer of cells with serial dilutions oftest samples and overlaying with a viscous suspension of the polymerAvicel (FMC Biopolymers). Plaques were allowed to develop over a periodof 48 hours @ 35° C., formalin fixed, stained with crystal violet andvisualized (FIG. 4). The plaque count was used to calculate infectiousviral titers in the test samples. The relative number of viable cellsfollowing infection was used as a measure of CPE. Sub-confluent cellcultures were exposed to a compound/virus mix (moi=1.0) for a period ofone hour @ 35° C. Unbound virus and drug were then removed and replacedwith virus growth medium. Cell viability was determined following 48hours incubation using Promega's CellTiter Blue reagents (resazurin) asthe extent of the metabolic conversion of the non-fluorescent resazurinto fluorescent resorufin read (555/585 nm excitation/emission;SpectraMax M2; Molecular Probes).

From these studies, we have found that the exemplary antibody inhibitsvirus-induced plaque production.

Example 5. IC₅₀ Evaluation of an Influenza Antibody

The present example illustrates the ability of an exemplary influenzaantibody to prevent virus infectivity in an in vitro binding assays.

The IC₅₀s for anti-influenza agents targeting HA have previously beenquantified. Studies utilized the H1N1 influenza strain PR8 (A/PuertoRico/8/34). In brief, confluent MDCK cells were infected with PR8 [4E3PFU/mL] and pre-incubated for 40 minutes with varying concentrations ofanti-influenza agents. After one hour of infection, media was removedand replaced with virus-free, drug-containing media or virus-free,drug-free media depending upon the experiment. After 48 hours ofincubation at 37° C., 5% CO₂, supernatants were collected. Viral RNA wasisolated, and viral titer was quantified by real time PCR using primersspecific for the virus M protein.

Initial assessment of IC₅₀ values for an exemplary antibody of Example1, was determined by microneutralization assay followed by quantitativePCR (qPCR). Mixtures of virus (PR8) and influenza targeting antibody atvarious titers and concentrations, respectively, were pre-incubated for1 hour at 35° C. before being applied to MDCK cell cultures in a 96-welltissue culture plate (˜10,000 cells/well). After an additional 48 hoursof incubation, the culture medium was collected from each well for viralyield.

Media from triplicate samples were combined and then subjected to directquantification of viral yield by qPCR. Viral titers were calculated fromthe PCR Ct values with the aid of an internal standard curve, and theIC₅₀ values were determined by plotting the calculated titers againstantibody concentrations (FIG. 5). The results showed 50% inhibition(IC₅₀) of PR8 viral particles with 0.5 μg/ml of the antibody fromexample 1. These results suggest that the antibody of Example 1 is ableto inhibit influenza virus infectivity. In some embodiments, we haveobserved IC₅₀ values in the range of 0.01 to 10 μg/ml for inhibitinginfluenza infectivity for various influenza strains. In someembodiments, we have observed IC₅₀ values in the range of 0.1 to 100μg/ml for inhibiting influenza infectivity for various influenzastrains.

Example 6. Influenza Antibody Binds HA Polypeptides In Vivo

The present example illustrates the ability of influenza antibodies tobind HA polypeptides in vivo.

The ability of the exemplary influenza antibody of Example 1 to inhibitinfluenza infection was evaluated in vivo. More particularly, assayswere performed to evaluate whether antibody administered at variousconcentrations prior to infection, could serve as a prophylaxis inreducing influenza infectivity.

BALB/c mice (4-6 weeks old) were procured from Charles River Labs. Micewere weighed and divided into four groups of 6 mice each for theexperiment. Groups consisted of: Group 1—no treatment control; Group2—treatment with antiviral drug on days −1, 0 and 1; Group 3—treatmentwith single dose of 6 mg/kg of antibody; and Group 4—treatment with asingle dose of 10 mg/kg of antibody. On the first day prior to infection(day −1) each group was administered with isoflurane, and dosed withcontrol, 75 mg/kg of antiviral drug Ribavirin and influenza antibody(either 6 or 10 mg/kg) and allowed to recover (<2 min). The followingday (day 0) each group was re-administered with isoflurane andchallenged intranasally with a lethal dose of H1N1 PR8 virus. Asindicated above, Group 2 was also administered 75 mg/kg on days 0 and 1of the experiment. Mice were monitored daily for 14 days for changes inweight loss associated with viral infection, and survival rate recordeddaily. Clinical signs of influenza infection in mice include hunchedposture, ruffled fur, rapid breathing, loss of appetite, weight loss,and death. FIG. 6 demonstrates that those mice prophylactically treatedwith antibody demonstrated little to no weight loss, when compared tothe control group.

In addition to weight loss, viral yield was measured in a post-infectionbronchio-aveolar lavage assay. Nasal washes were collected on day 3 fromthree animals from each of groups 1, 2 and 4, and their lungs wereharvested prior to sacrifice. Bronchio-aveolar lavage fluid from thethree mice were combined and subjected to direct quantification of viralyield by qPCR. Viral RNA was isolated, and viral titer quantified byreal time PCR using primers specific for the virus M protein. Viraltiters were calculated from the PCR Ct values with the aid of aninternal standard curve. The data presented in FIG. 7 suggests that asingle prophylactic antibody treatment prior to influenza infectionleads to a reduced level of infection (as demonstrated by viral load)similar to that of the antiviral drug Ribavirin.

The results of these studies show, among other things, that providedantibodies can successfully delay and/or prevent onset of H1N1 infectionin mice, when administered prior to infection.

Example 7. Evaluation of Influenza Antibodies as a Therapy In Vivo

The present example illustrates the ability of influenza antibodies tobind HA polypeptides in vivo for use as a treatment.

The ability of the exemplary influenza antibody of Example 1 to inhibitinfluenza infection was evaluated in vivo. Assays were performed toevaluate whether antibody administered at various concentrationspost-infection, could serve as treatment therapy in reducing influenzainfectivity.

BALB/c mice (4-6 weeks old) were procured from Charles River Labs. Micewere weighed and divided into three groups of 6 mice each for theexperiment. Groups consisted of: Group 1—no treatment control; Group2—treatment with a single dose of 5 mg/kg of antibody; and Group3—treatment with a single dose of 10 mg/kg of antibody. At the start ofthe experiment (day 0), each group is administered isoflurane andchallenged intranasally with a lethal dose of H1N1 PR8 virus. Two daypost-infection (day 2), each group was re-administered isoflurane, anddosed with control, 5 mg/kg of influenza antibody, or 10 mg/kg ofinfluenza antibody and allowed to recover (<2 min). Mice were monitoreddaily for 14 days for changes in weight loss associated with viralinfection, and survival rate recorded daily. Clinical signs of influenzainfection in mice include hunched posture, ruffled fur, rapid breathing,loss of appetite, weight loss, and death. FIG. 8 demonstrates that thosemice treated with antibody therapy showed reversal of one or moresymptoms of infection. For example, a reversal in viral associatedweight loss, resulting in an increased survival rate when compared tocontrol. These results indicate that antibody therapy can reversedisease state and symptoms associated with influenza infection.

Example 8. Pharmacokinetic Evaluation of an Antibody In Vivo

The present Example describes pharmacokinetic properties of an influenzaantibody in vivo.

BALB/c mice (4-6 weeks old) were procured from Charles River Labs andplaced in a single group of 6 mice for the experiment. Each mousereceived a single bolus injection of 5 mg/ml of antibody. Serum sampleswere collected at predetermined time-points over a 180 hr period.Collected samples were evaluated by ELISA, using methods as describedherein. Briefly, maxisorp 96-well plate wells were coated with 0.2 μg ofhuman IgG and left overnight at 4° C. The human IgG coated plates werewashed thrice with PBS and blocked with 1% BSA in PBST. Serum samplescollected over the 180 hr. period following antibody injection, wereadded to the human IgG coated wells and the plate was incubated at RTfor 2 hrs. The plate was washed thrice with PBST and the wells wereincubated with goat-anti-mouse HRP antibody for 1 hr at RT.Post-incubation the wells were washed with PBST and the bound HRP wasmeasured using TMB substrate. TMB substrate was added to the wells,incubated for 3 minutes, followed by addition of 1 N sulfuric acid.Absorbance was measured at 450 nm. The data in FIG. 9 demonstrates arapid distribution phase from 0 to 30 hours, with a peak serumconcentration at approximately 30 hours. FIG. 9 also demonstrates agradual elimination phase from 30 to 160 hours, indicating a reductionin serum antibody concentration, which suggests possible clearance orpartitioning of the antibody into different compartments of the mouse'sbody.

Example 9. Binding Agents in Diagnostics

The present example illustrates the ability of exemplary influenzaantibodies to provide a rapid way for (a) identifying the presence ofinfluenza virus in a biological sample and (b) characterizing the virus,based on the subtype.

A sandwich ELISA (virus typing ELISA assay) assay is used for thepurpose of identifying the presence of influenza virus andcharacterizing the virus subtype. For the virus typing ELISA assay,96-well plates will be coated with 2 μg of influenza antibody andincubated overnight at 4° C. The plates would then be extensively washedwith PBS and blocked with 1% BSA in PBST for 1 hr. Post blocking, theplates are washed with PBST and stored at 4° C. till further use.

Biological samples suspected of containing influenza virus are dilutedin sample buffer (PBS) either directly or post processing. The dilutedsamples are applied to the influenza antibody-coated wells and incubatedfor 2 hrs at room temperature (RT) followed by extensive washing. Virusfrom the sample are thus captured by the influenza antibody and lenditself for further analysis. Subtype specific antibody are applied todifferent wells for 1 hr at RT. After further washes with PBST,HRP-conjugated secondary antibodies are applied to the wells.Post-incubation, the wells are washed, and treated with TMB substrateand 1 N sulfuric acid. The absorbance at 450 nm is measured using aspectrophotometer. Appropriate negative and positive controls areincluded.

The results of the virus typing ELISA assay yield information about thepresence of influenza virus in a sample and the subtype of the virus.

Example 10. Antibodies for Influenza Virus Glycan Characterization

The present example illustrates the ability of an exemplary influenzaantibody as means to enrich and label influenza virus for glycancharacterization using a glycan typing assay.

In the glycan typing assay, the influenza antibody is conjugated to Qdot525 Carboxyl Quantum Dots using EDC chemistry as per manufacturersinstruction. The Qdot-influenza antibody complex is added to processedbiological samples and stirred well for 2 hrs. The sample is thencentrifuged and the Qdot-influenza antibody complex is washed thricewith PBST. This complex is then applied on glycan array (containingumbrella and cone topology glycans). After incubating for 2 hrs at RT,the wells are washed thrice with PBST. The bound fluorescence ismeasured using SpectraMax M2e spectrophotometer using bottom read mode.

The results of this assay yield information regarding the glycancharacterization of influenza viruses.

Example 11. Minimum Inhibitory Activity Assay

A method to determine the minimum inhibitory concentration (MIC) of theinfluenza antibody against influenza A is used. To be active in thisassay, the influenza antibody must bind to the virus and neutralize thevirus' ability to form plaques. Briefly, an influenza antibody isserially diluted in two fold increments in PBS to form a concentrationgradient across multiple wells. A known number of viral plaque formingunits are added to each well and after 1 hour incubation, the mixture isadded to an MDCK monolayer to allow viral binding. An Avicel overlayencourages plaque formation and the plaques are visualized byimmunostain. The lowest concentration of agent to prevent plaqueformation is reported as the MIC. These studies utilize H1N1 strain PR8(A/Puerto Rico/8/34). Representative influenza antibody activity in theassay versus H1N1 is determined.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

We claim:
 1. A method of treating a subject, the method comprising stepsof: administering to a subject suffering from or susceptible toinfluenza infection a therapeutically effective amount of an antibodythat comprises a heavy chain having an amino acid sequence that is atleast 95% identical to SEQ ID NO:1 and a light chain having an aminoacid sequence that is at least 95% identical to one of SEQ ID NOs:33-43.2. The method of claim 1, wherein the antibody is formulated foradministration by a route selected from the group consisting of oral,intravenous, intramuscular, intra-arterial, subcutaneous,intraventricular, transdermal, interdermal, rectal, intravaginal,intraperitoneal, topical, mucosal, nasal, buccal, enteral, sublingual,intratracheal or bronchial.
 3. The method of claim 2, wherein theadministration is by intratracheal or bronchial instillation.
 4. Themethod of claim 2, wherein the administration is by inhalation.
 5. Themethod of claim 1, wherein the antibody is formulated as an oral spray,a nasal spray, or an aerosol.
 6. A method of detecting a hemagglutinin(HA) polypeptide in a sample, the method comprising steps of: contactinga sample with an antibody that comprises a heavy chain having an aminoacid sequence that is at least 95% identical to SEQ ID NO:1 and a lightchain having an amino acid sequence that is at least 95% identical toone of SEQ ID NO:33-43; and detecting binding in the sample.
 7. Themethod of claim 6, wherein the sample is or comprises one or more ofblood, serum/plasma, peripheral blood mononuclear cells/peripheral bloodlymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermallesion swabs, cerebrospinal fluids, cervical smears, pus samples, foodmatrices, and tissues from various parts of the body such as brain,spleen, and liver.
 8. The method of 6, wherein the sample is anenvironmental sample.
 9. The method of claim 8, wherein theenvironmental sample is or comprises one or more of soil, water andflora.